Compounds for inducing anti-tumor immunity and methods thereof

ABSTRACT

Described herein is a previously unknown function of XBP1 in controlling anti-tumor immunity. It is shown that inhibiting XBP1 in tumor-associated dendritic cells inhibits tumor growth and induces protective anti-tumor immune responses.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/024,215, filed Mar. 23, 2016, which is a National Stage applicationclaiming priority to PCT/US2014/057525, filed Sep. 25, 2014, whichapplication claims the benefit of U.S. Provisional Patent ApplicationSer. No. 61/882,461, filed Sep. 25, 2013, where the entire content ofeach of the above-referenced patent applications is incorporated byreference as if fully set forth herein.

BACKGROUND OF THE INVENTION

The Endoplasmic Reticulum (ER) functions primarily to process newlysynthesized secretory and transmembrane proteins. However, abnormalaccumulation of unfolded proteins in this compartment causes a state of“ER stress”, which is a hallmark feature of secretory cells and manydiseases, including diabetes, neurodegeneration and cancer (Hetz et al.,Nature Reviews Drug Discover 2013; 12, 703-719). Adaptation toprotein-folding stress is mediated by the activation of an integratedsignal transduction pathway known as the ER stress response, or theunfolded protein response (UPR). This coordinated pathway signalsthrough three distinct stress sensors located at the ER membrane:IRE-1α, ATF6, and PERK (Hetz et al., Nature Reviews Drug Discover 2013;12, 703-719). The most conserved arm of the UPR involves IRE-1α. DuringER stress, this kinase oligomerizes, autophosphorylates, and uses itsendoribonuclease activity to excise a 26-nucleotide fragment from theunspliced XBP1 mRNA (Yoshida et al., Cell 2001; 107; 881-891). Theseevents give rise to functional XBP1, a potent multitasking transcriptionfactor that promotes the expression of ER chaperones and regulatesdistinct sets of target genes in a cell type-specific manner(Acosta-Alvear et al., Mol Cell 2007; 27, 53-66; Lee et al., Mol CellBiol 2003; 23, 7448-7459; Yoshida et al., Cell 2001; 107; 881-891).Importantly, while XBP1 has been shown to control the maintenance ofvarious immune cells under non-pathological conditions, a role for thistranscription factor as a negative regulator of anti-tumor responses andnormal immune function in cancer has never been reported.

Aggressive tumors have evolved strategies to thrive in adverseconditions such as hypoxia, nutrient starvation and high metabolicdemand. Cancer cells constantly undergo ER stress, but they ensuresurvival by adjusting the ER protein folding capacity via the UPR (Hetzet al., Nature Reviews Drug Discover 2013; 12, 703-719). In malignantcells, XBP1 confers drug resistance by preventing drug-inducedcell-cycle arrest and mitochondrial permeability and apoptosis (Gomez etal., FASEB J 2007; 21, 4013-4027). XBP1 drives the pathogenesis ofmultiple myeloma (Carrasco et al., Cancer Cell 2007; 11, 349-360; Lee etal., Proc Natl Acad Sci USA 2003; 100, 9446-9951) and of chroniclymphocytic leukemia (Sriburi et al., J Cell Biol 2004; 167, 35-41).Further, it was recently demonstrated that XBP1 fosters triple-negativebreast cancer progression by promoting tumor cell survival andmetastatic capacity under hypoxic conditions (Chen et al., Nature 2014;508, 103-107). While XBP1 expression in cancer cells has been shown todirectly support tumorigenesis, the role of this ER stress sensor insculpting a tumor-permissive immune milieu has not been established.

In most solid cancers, nonmalignant cells such as leukocytes, vascularcells and fibroblasts, stimulate tumor development and progression(Bhowmick et al., Nature 2004; 432, 332-337; Whiteside, Oncogene 2008;27, 5904-5912). Leukocyte recruitment to established cancers results indiverse pro-tumoral effects including the secretion of growth factorsthat enhance tumor cell proliferation and metastasis (Coussens et al.,Cell 2000; 103, 481-490; Coussens and Werb, Nature 2002; 420, 860-867;Mantovani et al., Nature 2008; 454, 436-444); the induction of tumorvascularization via paracrine mechanisms (De Palma et al., TrendsImmunol 2007; 28, 519-524); and the orchestration of immunosuppressivenetworks (Zou, Nat Rev Cancer 2005; 5, 263-274) that restrain theprotective role of the scarce leukocyte subsets with inherent anti-tumorcapacity. Ovarian tumors subvert the normal activity of infiltratingdendritic cells (DCs) to inhibit the function of otherwise protectiveanti-tumor T cells (Cubillos-Ruiz et al., J Clin Invest 2009; 119,2231-2244; Curiel et al., Nat Med 2003; 9, 562-567; Huarte et al.,Cancer Res 2008; 68, 7684-7691; Scarlett et al., Cancer Res 2009; 69,7329-7337; Scarlett et al., J Exp Med 2012). Eliminating or“re-programming” tumor-associated DCs (tDCs) in vivo has beendemonstrated to abrogate ovarian cancer progression (Cubillos-Ruiz etal., J Clin Invest 2009; 119, 2231-2244; Curiel et al., Nat Med 2003; 9,562-567; Huarte et al., Cancer Res 2008; 68, 7684-7691; Nesbeth et al.,Cancer Res 2009; 69, 6331-6338; Nesbeth et al., J Immunol 2010; 184,5654-5662; Scarlett et al., Cancer Res 2009; 69, 7329-7337; Scarlett etal., J Exp Med 2012), but the precise molecular pathways that tumorsexploit in DCs to co-opt their normal activity remain poorly understood,and therefore available therapeutics are limited.

SUMMARY OF THE INVENTION

Dendritic cells (DCs) are required to initiate and sustain anti-cancerimmunity. However, tumors efficiently manipulate DC function to evadeimmune control. The Endoplasmic Reticulum (ER) stress response has beenshown to operate in malignant cells to support tumor growth, but priorto the discovery of the present invention, its role in sculpting theimmune response to cancer was elusive. The present invention is based,at least in part, on the new finding that constitutive activation of theER stress sensor XBP1 in tumor-associated DCs (tDCs) drives ovariancancer progression. Here, it is reported that, XBP1-deficient tDCsshowed reduced intracellular lipid accumulation and demonstratedimproved antigen-presenting capacity, leading to enhanced intra-tumoralT cell activation and increased host survival. The present invention isalso based, at least in part, on the new finding that therapeutic XBP1silencing using siRNA-loaded nanoparticles restored proper tDC functionin situ and extended host survival by inducing protective anti-tumorimmunity. In particular, the instant inventors have discovered thattumors rely on DC-intrinsic XBP1 to elude immune control. Thesefindings, for the first time, reveal a key function for XBP1 inanti-tumor immunity, opening new avenues for therapeutics that targetthe ER stress response. Such therapeutics could potentially inhibittumor growth directly while simultaneously inducing robust anti-tumorimmunity.

In one embodiment, the present invention pertains to the unexpecteddiscovery that the ER stress sensor, XBP1 functions as a crucial driverof DC dysfunction in the tumor microenvironment. In another embodiment,the present invention provides that the ER stress sensor XBP1 isconstitutively active in ovarian cancer associated dendritic cells(DCs). In another embodiment, the present invention provides that thelipid peroxidation byproduct 4-HNE triggers ER stress and XBP1activation in DCs. In another embodiment, the present invention providesthat XBP1 regulates lipid metabolism and antigen presentation bytumor-associated DCs. In other embodiments, the invention provides thattargeting XBP1 in tumor-associated DCs (tDCs) extends host survival byenhancing anti-tumor immunity.

Accordingly, in one aspect, the invention pertains to a method forenhancing or inducing an anti-tumor immune response in a subject,including administering to the subject an effective amount of a director indirect inhibitor of XBP1, thereby enhancing or inducing theanti-tumor immune response in the subject.

In another aspect, the invention pertains to a method for treating orreducing the progression of ovarian cancer in a subject, includingadministering to the subject an effective amount of a direct or indirectinhibitor of XBP1, thereby treating or reducing the progression ofovarian cancer in the subject.

In another aspect, the invention pertains to a method for inducing,enhancing or promoting the immune response of cancer-associateddendritic cells in a subject including administering to the subject aneffective amount of a direct or indirect inhibitor of XBP1, therebyinducing, enhancing or promoting the immune response ofcancer-associated dendritic cells in the subject.

In one embodiment, the subject has ovarian cancer. In anotherembodiment, the subject has primary ovarian cancer or metastatic ovariancancer. In another embodiment, the subject has a carcinoma, anadenocarcinoma, an epithelial cancer, a germ cell cancer or a stromaltumor.

In another embodiment, the anti-tumor immune response is induced orenhanced in T cells in the subject. For example, intra-tumoral T cellsmay be activated in the subject. In certain embodiments, the anti-tumorimmune response may be antigen presentation by T cells. In otherembodiments, the anti-tumor immune response may be an immunogenicfunction.

In one embodiment, the inhibitor of XBP1 is a direct inhibitor. Inanother embodiment, the inhibitor of XBP1 is an indirect inhibitor.

For example, the inhibitor of XBP1 may be a nucleic acid molecule thatis antisense to an XBP1-encoding nucleic acid molecule, an XBP1 shRNA,and XBP1 siRNA, a microRNA that targets XBP1, ananoparticle-encapsulated XBP1 siRNA, an XBP1 siRNA-PEI nanoparticle, adominant negative XBP1 molecule, an XBP1-specific antibody or a smallmolecule inhibitor of XBP1.

In certain embodiments, the inhibitor of XBP1 is an agent that inhibitsIRE-1α or an agent that inhibits the generation of functional XBP1. Forexample, the inhibitor of IRE-1a may be an IRE-1a shRNA, an IRE-1asiRNA, or a nanoparticle-encapsulated IRE-1a siRNA. For example, anagent that inhibits the generation of functional XBP1 may be an agentthat inhibits an endonuclease that produces functional XBP1.

In one embodiment, the inhibitor of XBP1 is administered systemically,parenterally, or at tumor locations in the subject. For example, theinhibitor of XBP1 may be administered at the site of the ovarian canceror ovarian tumor.

In another embodiment, the inhibitor of XBP1 targets tumor-associateddendritic cells (tDCs)

In certain embodiments, the inhibitor of XBP1 is administered incombination with a second cancer therapeutic agent. For example, theinhibitor of XBP1 may be administered in combination with achemotherapeutic agent.

In another embodiment, treatment of the subject with a direct orindirect inhibitor of XBP1 induces extended survival of the subject.

In another aspect, the invention pertains to a method for enhancing orinducing an anti-tumor immune response in a subject includingadministering to the subject an effective amount of a direct or indirectinhibitor of IRE-1α, thereby enhancing or inducing the anti-tumorresponse in the subject.

In one embodiment, the subject has ovarian cancer. In anotherembodiment, the subject has a carcinoma, an adenocarcinoma, anepithelial cancer, a germ cell cancer or a stromal tumor.

In another embodiment, the anti-tumor immune response is induced orenhanced in T cells in the subject. For example, intra-tumoral T cellsmay be activated in the subject.

In one embodiment, the inhibitor of IRE-1α is a direct inhibitor. Inanother embodiment, the inhibitor of IRE-1α is an indirect inhibitor.

For example, the inhibitor of IRE-1α may be a nucleic acid molecule thatis antisense to an IRE-1α-encoding nucleic acid molecule, an IRE-1αshRNA, and IRE1α siRNA, a microRNA that targets IRE-1α, ananoparticle-encapsulated IRE-1α siRNA, an IRE-1α siRNA-PEInanoparticle, a dominant negative IRE-1α molecule, an IRE-1α-specificantibody and a small molecule inhibitor of IRE-1α.

In one embodiment, the inhibitor of IRE-1α is administered systemically,parenterally, or at tumor locations in the subject. For example, theinhibitor of IRE-1α may be administered at the site of the ovariancancer or ovarian tumor.

In another embodiment, the inhibitor of IRE-1α targets tumor-associateddendritic cells (tDCs)

In certain embodiments, the inhibitor of IRE-1α is administered incombination with a second cancer therapeutic agent. For example, theinhibitor of IRE-1α may be administered in combination with achemotherapeutic agent.

In another embodiment, treatment of the subject with a direct orindirect inhibitor of IRE-1α induces extended survival of the subject.

In another aspect, the invention pertains to a method for activating orenhancing Type 1 immunity in ovarian cancer-infiltrating T cells in asubject including administering to the subject an effective amount of adirect or indirect inhibitor of XBP1, thereby activating or enhancingType 1 immunity in ovarian cancer-infiltrating T cells in the subject.

In yet another aspect, the invention pertains to a method for reducingER stress in tumor-associated dendritic cells (tDCs) in a subjectincluding administering to the subject an effective amount of a director indirect inhibitor of 4-HNE to inhibit XBP1 activation, therebyreducing ER stress in tumor-associated dendritic cells in the subject.

In a further aspect, the invention pertains to a method for reducing orpreventing intracellular lipid accumulation in tumor-associateddendritic cells (DCs) including administering to the subject aneffective amount of a direct or indirect inhibitor of XBP1, therebyreducing or preventing intracellular lipid accumulation intumor-associated dendritic cells in the subject.

In one embodiment, the number of cytosolic lipid droplets in thedendritic cell is reduced. In another embodiment, the intracellularlevels of total triglycerides is reduced.

In another aspect, the invention pertains to a method for enhancing orinducing T cell activation at a tumor site in a subject includingadministering to the subject an effective amount of a direct or indirectinhibitor of XBP1, thereby enhancing or incuding T cell activation atthe tumor site in the subject.

In yet another aspect, the invention pertains to a method for inducing,enhancing or promoting the antigen presenting capacity oftumor-associated dendritic cells (DCs) in a subject includingadministering to the subject an effective amount of a direct or indirectinhibitor of XBP1, thereby inducing, enhancing or promoting the antigenpresenting capacity of tumor-associated dendritic cells in the subject.

In yet another aspect, the invention pertains to a method for inducing,enhancing or promoting the antigen presenting capacity oftumor-associated dendritic cells (tDCs) in a subject includingcontacting dendritic cells with an effective amount of a direct orindirect inhibitor of XBP1 and then administering the dendritic cells tothe subject, thereby inducing, enhancing or promoting the antigenpresenting capacity of tumor-associated dendritic cells in the subject.

In a further aspect, the invention pertains to a method of identifying acompound useful in enhancing or inducing anti-tumor immunity in asubject, including providing an indicator composition comprising XBP1,or biologically active portions thereof; contacting the indicatorcomposition with each member of a library of test compounds; selectingfrom the library of test compounds a compound of interest that interactswith XBP1, or biologically active portions thereof; and contactingovarian cancer cells with the compound of interest, wherein the abilityof the compound to enhance or induce anti-tumor immunity in the subjectis indicated by the ability of the compound to inhibit growth of ovariancancer cells as compared to the growth of ovarian cancer cells in theabsence of the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F: Shows the gating strategy used for FACS analysis

FIG. 1A: depicts the gating strategy used to isolate human ovariancancer-associated DCs present in solid tumors.

FIG. 1B: depicts the gating strategy used to isolate human ovariancancer-associated DCs present in malignant ascites specimens.

FIG. 1C: depicts the gating strategy used to isolate tDCs fromp53-Kras-driven primary ovarian tumors of mice bearing advancedmetastatic ID8-Defb29-VegfA ovarian tumors.

FIG. 1D: depicts the gating strategy used to isolate tDCs from malignantovarian ascites of mice bearing advanced metastatic ID8-Defb29-VegfAovarian tumors.

FIG. 1E: graphically depicts robust expression of Clec9A/DNGR-1 andZbtb46 by murine CD45⁺CD11c⁺MHC-II⁺CD11b⁺CD8α⁻ tumor-infiltratingmyeloid cells, reinforcing their identity as genuine classical DCs(cDCs). These two markers distinguish cDCs and their committedprogenitors from other closely related mononuclear phagocytic lineages.

FIG. 1F: depicts the gating strategy to isolate controlCD45⁺CD11c⁺MHC-II⁺CD11b⁺CD8α⁻ DCs from the spleen (sDCs).

FIG. 2A-F: Shows constitutive XBP1 activation in human and mouse ovariancancer-associated DCs.

FIG. 2A: depicts PCR analysis of Xbp1 mRNA splicing in ovarian cancertDCs from human (1-12) and mouse (p53/K-ras and ID8-Def29/Vegf-A)origin. XBP1u, unspliced form; XBP1s, spliced form; Actb, β-actin.

FIG. 2B: graphically depicts expression of total XBP1 mRNA (Left) andspliced XBP1 (XBP1s) mRNA (Right). Expression of the indicatedtranscript was determined by RT-qPCR. sDC, splenic DCs.

FIG. 2C: depicts a western blot of XBP1s protein expression in nuclearextracts obtained from the indicated DCs. Lamin B was used as loadingcontrol. In all cases data are representative of 3 independentexperiments with similar results.

FIG. 2D: graphically depicts expression of ERdj4 mRNA (Left) and Sec61a1mRNA (Right). Expression of the indicated transcript was determined byRT-qPCR.

FIG. 2E: graphically depicts expression of BiP mRNA (Left) and CHOP mRNA(Right). Expression of the indicated transcript was determined byRT-qPCR.

FIG. 2F: graphically depicts the expression of CHOP in tDCs sorted fromhuman patient ovarian cancer specimens (determined by RT-qPCR)correlated with the percentage of CD45⁺CD3⁺ T cells present in eachindividual tumor (circle) or ascites (square) sample. r, Spearman's Rankcorrelation coefficient.

FIG. 3A-F: Shows the effect of diverse cytokines and hypoxia mimickingconditions on XBP1 activation by DCs

FIGS. 3A-D: graphically depict the effect of diverse cytokines on XBP1activation by DCs. Splicing and upregulation of XBP1 was determined byRT-qPCR analysis.

FIGS. 3E-F: graphically depict the effect of hypoxia-mimickingconditions on XBP1 activation by DCs.

FIG. 4A-J: Provides lipid peroxidation byproduct 4-HNE triggers ERstress in DCs.

FIG. 4A: depicts increased intracellular lipid content in tDCs comparedwith control sDCs from naïve or tumor-bearing mice (Ovca).Representative FACS analysis of lipid staining for DCs from theindicated sources (Left). Intracellular lipid quantification expressedas mean fluorescence intensity (MFI) of Bodipy 493/503 staining (Right).

FIG. 4B: graphically depicts the quantification of intracellular ROSlevels in DCs expressed as geometric MFI (gMFI) of DCFDA staining.

FIG. 4C: graphically depicts the levels of 4-HNE-protein adducts incell-free malignant ascites obtained from hosts bearing metastaticovarian cancer.

FIG. 4D: graphically depicts the quantification of total intracellular4-HNE-protein adducts in tDCs isolated from metastatic ovarian cancerascites samples of human and mouse origin.

FIG. 4E: graphically depicts the intracellular levels of 4-HNE-proteinadducts in tDCs isolated from mice with advanced ID8-Defb29-VegfAovarian tumors and exposed to cell-free malignant ascites in thepresence or absence of Vitamin E (VitE).

FIG. 4F: graphically depicts the rapid generation of intracellular4-HNE-protein adducts in naïve sDCs incubated with increasingconcentrations of purified 4-HNE.

FIGS. 4G-H: depict the quantification of XBP1s gene expression in sDCsexposed for 3h to increasing amounts of purified 4-HNE. Data arerepresentative of at least two independent experiments with similarresults.

FIG. 4I: depict the quantification of total XBP1 (left) and ERdj4(right) gene expression in sDCs exposed for 3h to increasing amounts ofpurified 4-HNE. Data are representative of at least two independentexperiments with similar results.

FIG. 4J: depict the quantification of total BiP (left) and CHOP (right)gene expression in sDCs exposed for 3h to increasing amounts of purified4-HNE. Data are representative of at least two independent experimentswith similar results.

FIG. 5A-C: Shows the efficient and selective XBP1 deletion in DCs viaCD11c-controlled Cre expression.

FIG. 5A: depicts a Schematic of Xbp1 exon 2 deletion.

FIG. 5B: graphically depicts deletion efficiency of XBP1, which wasdetermined by RT-qPCR using primers that selectively amplify exon 2 ofXbp1 (see methods).

FIG. 5C: depicts induction of canonical XBP1 target genes ERdj4 (Left)and Sec61 (Right) upon stimulation (determined via RT-qPCR). Data werenormalized to endogenous Actb expression in each sample. Data arerepresentative of at least three independent experiments with similarresults.

FIG. 6A-I: Shows the assessment of immune cell population in conditionalknockout mice

FIGS. 6A-C: depict representative FACS analysis of DC populations in thespleen.

FIG. 6D-E: depict representative FACS analysis of DC populations ininfiltrating p53/K-ras ovarian tumors.

FIG. 6F-G: depict representative FACS analysis of DC populations inmalignant ID8-Defb29/Vegf-A ovarian cancer ascites of XBP1^(f/f) (top)or XBP^(f/f) CD11c-Cre (bottom) mice.

FIG. 6H-I: depicts the proportions (FIG. 6H) and absolute numbers (FIG.6I) of depicted immune cell populations in the spleen of wild type orconditional knockout mice. pDC, plasmacytoid DCs. Data arerepresentative of 3 mice per group. *P<0.05

FIG. 7A-I: Shows ovarian cancer progression in hosts lacking XBP1 inDCs.

FIG. 7A: depicts p53/K-ras-driven ovarian tumors generated in hostsreconstituted with bone marrow from either XBP1^(f/f) (top) orXBP1^(f/f) CD11c-Cre (bottom) donor mice as described in the methods andprimary tumors were resected 48 days after intrabursal injection ofCre-expressing adenovirus (ADV-Cre).

FIG. 7B: graphically depicts growth kinetics of p53/K-ras-driven ovariantumors in hosts reconstituted with bone marrow from the indicatedgenotypes.

FIG. 7C: graphically depicts the proportion of hosts presentingp53/K-ras-derived metastatic lesions in the peritoneal cavity 48 daysafter tumor induction.

FIG. 7D: depicts mice of the indicated genotypes implanted withID8-Defb29-VegfA ovarian cancer cells via intraperitoneal (i.p.)injection.

FIG. 7E: graphically depicts peritoneal metastases evaluated 3-4 weeksafter tumor implantation (n=3 mice per group). **P<0.001.

FIG. 7F: graphically depicts malignant ascites generation intumor-bearing mice expressed as percent weight gain due to progressiveaccumulation of peritoneal fluid. *P<0.05.

FIG. 7G: depicts reduced splenomegaly in tumor-bearing mice deficientfor XBP1 in CD11c⁺ DCs.

FIG. 7H: depicts overall survival rates in mice bearing aggressiveID8-Defb29-VegfA tumors. Data are representative of at least twoindependent experiments with similar results using 4-6 mice per group.**P<0.001.

FIG. 7I: depicts overall survival rates in mice bearing parental ID8tumors. Data are representative of at least two independent experimentswith similar results using 4-6 mice per group. **P<0.001.

FIG. 8A-D: Shows transcriptional analysis of ovarian cancer-associatedDCs devoid of XBP1.

FIG. 8A: depicts the top transcriptional regulators associated withdifferentially expressed gene signatures in wild type vs. XBP1-deficientDCs isolated from mice bearing ID8-Defb29/Vegf-A ovarian tumors for 3weeks.

FIG. 8B: depicts the main ER stress-related gene network controlled byXBP1 in tDCs based on Ingenuity Pathway Analysis (IPA).

FIG. 8C: depicts the expression levels of previously reported RIDDtargets in XBP1-deficient tDCs.

FIG. 8D: depicts the top significantly affected biological processesidentified in tDCs devoid of XBP1 (see methods).

FIG. 9A-G: Shows that XBP1 disrupts lipid homeostasis in ovariancancer-associated DCs.

FIG. 9A: depicts the downregulation of genes involved in the UPR/ERstress response in tDCs devoid of XBP1. WT, XBP1^(f/f) tDC. XBP1^(def),XBP1^(f/f) CD11c-Cre tDC (n=3/group). FDR, false discovery rate. RPKM,transcript abundance expressed as reads per kilobase per million reads.

FIG. 9B: depicts the downregulation of genes involved in the lipidmetabolism in tDCs devoid of XBP1. WT, XBP^(f/f) tDC. XBP1^(def),XBP1^(f/f) CD11c-Cre tDC (n=3/group). FDR, false discovery rate. RPKM,transcript abundance expressed as reads per kilobase per million reads.

FIG. 9C: depicts decreased intracellular lipid content in XBP1^(f/f)CD11c-Cre tDCs (n=3 mice per group) from mice bearing ID8-Defb29-VegfAtumors for 3 weeks evidenced by Bodipy493/503 staining.

FIG. 9D: depicts electron micrographs (12,000×) demonstrating largeintracellular lipid bodies in XBP1-sufficient, but not XBP1-deficienttDC.

FIG. 9E: graphically depicts quantification of lipid bodies in tDCssorted from mice bearing ID8-Defb29-VegfA ovarian tumors for 3-4 weeks.

FIG. 9F: graphically depicts quantification of intracellulartriglycerides (TAG) in tDCs sorted from mice bearing ID8-Defb29-VegfAovarian tumors for 3-4 weeks.

FIG. 9G: graphically depicts the intracellular lipid content of tDCsincubated in vitro with 25% cell-free ovarian cancer ascitessupernatants in the presence of the indicated inhibitors. Intracellularlipid content was assessed 24h later via Bodipy493/503 staining. Dataare representative of 3 independent experiments with similar results.#P<0.05 compared with control tDC incubated in the absence of cell-freeascites supernatants. *P<0.05 compared with ascites-exposed tDC but leftuntreated.

FIG. 10A-F: Shows ER stress and lipid accumulation in tDCs.

FIG. 10A: depicts increased expression of XBP1-controlled lipidbiosynthetic genes (Agpat6, Fasn, Lpar1) in tDCs isolated from micebearing ID8-Defb29-Vegf-A ovarian tumors for 4-5 weeks compared with theindicated control sDCs.

FIG. 10B: graphically depicts rapid upregulation of XBP1s, ERdj4 andXBP1-controlled lipid biosynthetic genes (Agpat6, Fasn, Lpar1) in naïvesDCs stimulated for 3h with increasing concentrations of purified 4-HNE.Data are normalized to endogenous levels of (β-actin in each sample, andare representative of three independent experiments with similarresults.

FIG. 10C: graphically depicts the lipidomic profile of tDCs obtainedfrom mice bearing ID8-Defb29/Vegf-A ovarian tumors for 4 weeks.

FIG. 10D: graphically depicts the lipidomic profile of cell-free ascitessupernatants obtained from mice bearing ID8-Defb29/Vegf-A ovarian tumorsfor 4 weeks.

FIG. 10E: graphically depicts relative expression of genes encodingscavenger receptors (Cd36, Cd68, Msr1) in XBP1-deficient vs. wild typetDCs.

FIG. 10F: depicts representative FACS analysis of Bodipy 493/503staining in tDCs exposed to cell-free ovarian cancer ascites in thepresence or absence of inhibitory compounds.

FIG. 11A-F: Shows that XBP1 promotes immune tolerance by DCs in theovarian cancer microenvironment.

FIG. 11A: shows a representative histogram analysis of surface moleculeexpression on tDCs from peritoneal wash samples of XBP1^(f/f) (striped)or XBP1^(f/f) CD11c-Cre (black) mice bearing ID8-Defb29-VegfA tumors for3-4 weeks. Dotted histograms indicate isotype control staining.

FIG. 11B: graphically depicts quantification of the data shown in FIG.5A expressed as geometric mean fluorescence intensity (gMFI) of staining(n=3 mice per group).

FIG. 11C: depicts CFSE-dilution analysis of OT-1 T cells cocultured withfull-length OVA-pulsed tDCs isolated from the peritoneal cavity ofXBP1^(f/f) or XBP1^(f/f) CD11c-Cre mice bearing ID8-Defb29-VegfA ovariantumors for 4 weeks (see methods for details).

FIG. 11D: graphically depicts the proportion of proliferating OT-1 Tcells described in FIG. 5C.

FIGS. 1E and 11F: depict enhanced endogenous T cell activation at tumorsites in mice devoid of XBP1 in CD11c⁺ tDCs. Peritoneal wash samplesfrom wild type (XBP1^(f/f), black bars) or XBP1-deficient (XBP1^(f/f)CD11c-Cre, gray bars) mice were collected 2-3 weeks after peritonealimplantation of ID8-Defb29-VegfA cancer cells. Surface expression ofCD44 and intracellular levels of tumoricidal IFN-γ were analyzed onCD3⁺CD4⁺ (FIG. 11E) or CD3⁺CD8⁺ (FIG. 11F) tumor-infiltrating T cells(TILs). In all cases, data are representative of at least twoindependent experiments using 3-4 mice group.

FIG. 12A-B: Shows antigen presentation by XBP1-deficient sDCs

FIG. 12A: depicts CFSE-dilution analysis of OT-1 T cells cocultured withfull length OVA-pulsed CD11c⁺MHC-II⁺CD11b⁺CD8α⁻ DCs isolated from thespleen (sDC) of XBP1^(f/f) or XBP1^(f/f) CD11c-Cre mice.

FIG. 12B: graphically depicts the proportion of proliferating OT-1 Tcells described in FIG. 12A. Data are representative of two independentexperiments with similar results.

FIG. 13A-C: shows biodistributin and silencing activity ofintraperitoneally injected siRNA-PEI nanoparticles

FIG. 13A: depicts the selective uptake of rhodamine-labelednanoparticles by CD11c⁺ tDC in the peritoneal cavity. Top, controluntreated mice. Bottom, representative data from mice receivingRhodamine-labeled siRNA-PEI nanocomplexes.

FIG. 13B-C: graphically depicts the biodistribution and silencingactivity of intraperitoneally injected siRNA-PEI nanoparticles.CD45⁺CD11c⁺Rhodamine⁺ tDCs were FACS-sorted from peritoneal wash samples3 days after nanoparticle injection and gene expression levels weredetermined via RT-qPCR. Data are representative of three independentsilencing experiments with 2-3 mice per group and all data werenormalized to endogenous Actb expression.

FIG. 14A-J: Shows therapeutic silencing of XBP1 improves tDC functionand triggers protective anti-tumor immunity.

FIG. 14A: shows representative CFSE dilution of OT-1 T cellsproliferating in vivo at tumor sites in full-length OVA-pulsed untreatedmice or after administration of immunostimulatory nanocomplexes carryingluciferase-matching (siLuc-PEI) or XBP1-specific (siXBP1-PEI) siRNA (seemethods for details).

FIG. 14B: graphically depicts the proportion of OT-1 T cells in theascites of treated mice (n=3/group) three days after transfer.

FIG. 14C: graphically depicts the division (Left) and proliferation(Right) index of transferred OT-1 T cells shown in FIG. 6B.

FIGS. 14D-H: show enhanced anti-tumor immune responses in mice treatedwith DC-targeting, XBP1-silencing nanocomplexes. ID8-Defb29/Vegf-Atumor-bearing mice (n=3/group) were treated at days 8, 13, 18 and 23post-tumor injection and peritoneal lavage samples were analyzed at day27. FIG. 14D graphically depicts the proportion of metastatic spheroidtumor cells (CD45-SSC^(hi)) found in the peritoneal cavity of treatedmice. FIG. 14E shows representative pictures of peritoneal lavagesobtained from treated mice. FIG. 14F graphically depicts the proportionof antigen-experienced (CD44⁺), activated (CD69⁺) CD4⁺ (Left) and CD8⁺(Right) T cells infiltrating tumor locations determined by FACS analyses(gated on CD3⁺ cells).

FIG. 14G-H show representative ELISA-based analysis showing increasedIFN-γ and Granzyme B secretion by peritoneal (FIG. 14G) and splenic(FIG. 14H) T cells isolated from mice treated with XBP1-silencingnanoparticles.

FIGS. 14I-J: depict overall survival rates in wild type (FIG. 14I) orRag2-deficient (FIG. 14J) ovarian cancer-bearing mice (n=6/group)treated with nanocomplexes at days 12, 16, 20, 24, 28 and 32 afterimplantation of ID8-Defb29/Vegf-A cancer cells. In all cases, data arerepresentative of at least two independent experiments with similarresults. ***P<0.001, Log-Rank Test.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogs of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al. Biol. Chem. 260:2605-2608, 1985); and Cassol et al, 1992;Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine andleucine, modifications at the second base can also be conservative. Theterm nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene. Polynucleotides used herein can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, the term “ER stress” refers to a perturbation in ERfunction and dysregulation of ER homeostasis due to an internal orexternal cellular insult. ER stress elicits a signaling cascade (i.e.,the unfolded protein response) to mitigate stress.

The term “Unfolded Protein Response” (UPR) or the “Unfolded ProteinResponse pathway” refers to an adaptive response to the accumulation ofunfolded proteins in the ER and includes the transcriptional activationof genes encoding chaperones and folding catalysts and protein degradingcomplexes as well as translational attenuation to limit furtheraccumulation of unfolded proteins. Both surface and secreted proteinsare synthesized in the endoplasmic reticulum (ER) where they need tofold and assemble prior to being transported.

As used herein, the term “dendritic cell” refers to a type ofspecialized antigen presenting cell (APC) involved in innate andadaptive immunity. Also referred to as “DC.” Dendritic cells may bepresent in the tumor microenvironment and these are referred to as“tumor-associated dendritic cells” or “tDCs.”

As used herein, the term “anti-tumor immunity” refers to an immuneresponse induced upon recognition of cancer antigens by immune cells.

As used herein, the term “T cell activation” refers to cellularactivation of resting T cells manifesting a variety of responses (Forexample, T cell proliferation, cytokine secretion and/or effectorfunction). T cell activation may be induced by stimulation of the T cellreceptor (TCR) with antigen/MHC complex.

As used herein, the term “antigen presenting capacity” refers to theability of antigen presenting cells (APCs) to present antigen to Tlymphocytes to elicit an immune response. In certain embodiments, theimmune response is a type I immunity response. In certain embodiments,the antigen presenting capacity is determined by measuring infiltrationand activation of T cells at tumor locations and/or secretion of IFN-γand Granzyme B ex vivo by APCs (i.e., dendritic cells).

As used herein, the term “anti-tumor T cells” refers to T lymphocytesthat have been activated by APCs, wherein the antigen is atumor-associated antigen. These T lymphocytes will subsequently inducethe killing of malignant cells.

As used herein, the term “anti-tumor response” refers to at least one ofthe following: tumor necrosis, tumor regression, tumor inflammation,tumor infiltration by activated T lymphocytes, or activation of tumorinfiltrating lymphocytes. In certain embodiments, activation oflymphocytes is due to presentation of a tumor-associated antigen byAPCs.

As used herein, the term “extended survival” refers to increasingoverall or progression free survival in a treated subject relative to anuntreated control.

As used herein, the term “test sample” is a sample isolated, obtained orderived from a subject, e.g., a human subject. The term “subject” or“host” is intended to include living organisms, but preferred subjectsor hosts are mammals, and in particular, humans or murines. The term“subject” or “host” also includes cells, such as prokaryotic oreukaryotic cells. In particularly preferred embodiments, the “testsample” is a sample isolated, obtained or derived from a subject, e.g.,a female human.

The term “sufficient amount” or “amount sufficient to” means an amountsufficient to produce a desired effect, e.g., an amount sufficient toreduce the size of a tumor.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, “combination therapy” embraces administration of eachagent or therapy in a sequential manner in a regiment that will providebeneficial effects of the combination and co-administration of theseagents or therapies in a substantially simultaneous manner. Combinationtherapy also includes combinations where individual elements may beadministered at different times and/or by different routes but which actin combination to provide a beneficial effect by co-action orpharmacokinetic and pharmacodynamics effect of each agent or tumortreatment approaches of the combination therapy. For example, the agentsor therapies may be administered simultaneously, sequentially, or in atreatment regimen in a predetermined order.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Various aspects described herein are described in further detail in thefollowing subsections.

ER Stress and UPR

The “Unfolded Protein Response” (UPR) or the “Unfolded Protein Responsepathway” is initiated when there is an accumulation of unfolded proteinsin the ER. This results in the transcriptional activation of genesencoding chaperones, folding catalysts, and protein degrading complexes,as well as translational attenuation to limit further accumulation ofunfolded proteins.

Since the ER and the nucleus are located in separate compartments of thecell, the unfolded protein signal must be sensed in the lumen of the ERand transferred across the ER membrane and be received by thetranscription machinery in the nucleus. The UPR performs this functionfor the cell. Activation of the UPR can be caused by treatment of cellswith reducing agents like DTT, by inhibitors of core glycosylation liketunicamycin or by Ca-ionophores that deplete the ER calcium stores.First discovered in yeast, the UPR has now been described in C. elegansas well as in mammalian cells. In mammals, the UPR signal cascade ismediated by three ER transmembrane proteins: the protein-kinase andsite-specific endoribonuclease IRE-1 alpha; the eukaryotic translationinitiation factor 2 kinase, PERK/PEK; and the transcriptional activatorATF6. If the UPR cannot adapt to the presence of unfolded proteins inthe ER, an apoptotic response is initiated leading to the activation ofJNK protein kinase and caspases 7, 12, and 3. The most proximal signalfrom the lumen of the ER is received by a transmembrane endoribonucleaseand kinase called IRE-1. Following ER stress, IRE-1 is essential forsurvival because it initiates splicing of the XBP1 mRNA, the splicedversion of which activates the UPR.

The unfolded protein response (UPR) is a major cellular stress responsepathway activated in tumors that allows them to adapt to the stresses ofthe tumor microenvironment. Similarly, the ER stress response has beenshown to operate in malignant cells to support tumor growth. The presentinvention provides the novel discovery that constitutive activation ofthe ER stress sensor, XBP1, in cancer-associated DCs drives cancerprogression (e.g., ovarian cancer progression). In certain embodiments,the ER stress response is dysregulated in cells present in the tumormicroenvironment. In certain embodiments, the ER stress response isactivated through signaling of the IRE-1/XBP1 pathway. In certainembodiments, the IRE-1/XBP1 pathway is constitutively active in cellspresent in the tumor microenvironment.

Accordingly, in one aspect, the invention pertains to a method fortreating or reducing the progression of ovarian cancer in a subject, themethod comprising administering to the subject a direct or indirectinhibitor of XBP1 or a direct or indirect inhibitor of IRE-1α such thatprogression of the ovarian cancer in the subject is inhibited.Non-limiting examples of direct inhibitors of XBP1 include a nucleicacid molecule that is antisense to an XBP1-encoding nucleic acidmolecule, an XBP1 shRNA, an XBP siRNA, a nanoparticle-encapsulated XBP1siRNA (e.g., polyethylenimine (PEI)-based nanoparticles encapsulatingsiRNA), a microRNA that targets XBP1, a dominant negative XBP1 molecule,an XBP1-specific antibody and small molecule inhibitors of XBP1.Non-limiting examples of indirect inhibitors of XBP1 include agents thattarget IRE-1, an endonuclease essential for proper splicing andactivation of XBP1, such that inhibition of IRE-1 leads to inhibition ofthe production of the spliced, active form of XBP1. Non-limitingexamples of IRE-1 inhibitors include a nucleic acid molecule that isantisense to an IRE-1-encoding nucleic acid molecule, an IRE-1 shRNA, anIRE-1 siRNA, a nanoparticle-encapsulated IRE-1 siRNA, a microRNA thattargets IRE-1, a dominant negative IRE-1 molecule, an IRE-1-specificantibody and small molecule inhibitors of IRE-1.

XBP1

X-box binding protein-1 (XBP1) is a transcription factor that acts as anER stress sensor by promoting the expression of ER chaperones andregulating distinct sets of target genes in a cell type-specific manner(Acosta-Alvear et al., 2007; Lee et al., 2003; Yoshida et al., 2001). Incertain embodiments, XBP1 is spliced via IRE-1 activation. In certainembodiments, spliced XBP1 enhances transcription of ER chaperones.

The X-box binding human protein (“XBP1”) is a DNA binding protein andhas an amino acid sequence as described in, for example, Liou, H. C.,et. al. 1990. Science 247, 1581-1584 and Yoshimura, T., et al. 1990.EMBO J. 9, 2537-2542, and other mammalian homologs thereof, such asdescribed in Kishimoto T., et al. 1996. Biochem. Biophys. Res. Commun.223, 746-751 (rat homologue). Exemplary proteins intended to beencompassed by the term “XBP1” include those having amino acid sequencesdisclosed in GenBank with accession numbers A36299 [gi: 105867],NP_005071 [gi:4827058], P17861 [gi: 139787], CAA39149 [gi:287645], andBAA82600 [gi:5596360] or e.g., encoded by nucleic acid molecules such asthose disclosed in GenBank with accession numbers AF027963[gi:13752783]; NM_013842 [gi: 13775155]; or M31627 [gi: 184485]. XBP1 isalso referred to in the art as TREB5 or HTF (Yoshimura, T., et al. 1990.EMBO Journal. 9, 2537; Matsuzaki, Y., et al. 1995. J. Biochem. 117,303). Like other members of the b-zip family, XBP1 has a basic regionthat mediates DNA-binding and an adjacent leucine zipper structure thatmediates protein dimerization.

There are two forms of XBP-1 protein, unspliced and spliced, whichdiffer in their sequence and activity. Unless the form is referred toexplicitly herein, the term “XBP1” as used herein includes both thespliced and unspliced forms. Spliced XBP1 (“XBP1s”) directly controlsthe activation of the UPR, while unspliced XBP1 functions to negativelyregulate spliced XBP1.

“Spliced XBP1” (“XBP1s”) refers to the spliced, processed form of themammalian XBP1 mRNA or the corresponding protein. Human and murine XBP1mRNA contain an open reading frame (ORF1) encoding bZIP proteins of 261and 267 amino acids, respectively. Both mRNA's also contain another ORF,ORF2, partially overlapping but not in frame with ORF1. ORF2 encodes 222amino acids in both human and murine cells. Human and murine ORF1 andORF2 in the XBP1 mRNA share 75% and 89% identity respectively. Inresponse to ER stress, XBP1 mRNA is processed by the ER transmembraneendoribonuclease and kinase IRE-1 which excises an intron from XBP1mRNA. In murine and human cells, a 26 nucleotide intron is excised. Theboundaries of the excised introns are encompassed in an RNA structurethat includes two loops of seven residues held in place by short stems.The RNA sequences 5′ to 3′ to the boundaries of the excised introns formextensive base-pair interactions. Splicing out of 26 nucleotides inmurine and human cells results in a frame shift at amino acid 165 (thenumbering of XBP1 amino acids herein is based on GenBank accessionnumber NM.sub.-013842 [gi:13775155] and one of skill in the art candetermine corresponding amino acid numbers for XBP1 from otherorganisms, e.g., by performing a simple alignment). This causes removalof the C-terminal 97 amino acids from the first open reading frame(ORF1) and addition of the 212 amino from ORF2 to the N-terminal 164amino acids of ORF1 containing the b-ZIP domain. In mammalian cells,this splicing event results in the conversion of a 267 amino acidunspliced XBP1 protein to a 371 amino acid spliced XBP1 protein. Thespliced XBP1 then translocates into the nucleus where it binds to itstarget sequences to induce their transcription.

“Unspliced XBP1” (“XBP1u”) refers to the unprocessed XBP1 mRNA or thecorresponding protein. As set forth above, unspliced murine XBP1 is 267amino acids in length and spliced murine XBP1 is 371 amino acids inlength. The sequence of unspliced XBP1 is known in the art and can befound, e.g., Liou, H. C., et. al. 1990. Science 247, 1581-1584 andYoshimura, T., et al. 1990. EMBO J. 9, 2537-2542, or at GenBankaccession numbers NM_005080 [gi: 14110394] or NM_013842 [gi: 13775155].

As used herein, the term “functional XBP1” refers to the spliced form ofXBP1, which acts as a transcription factor to activate the UPR.

As used herein, the term “ratio of spliced to unspliced XBP1” refers tothe amount of spliced XBP1 present in a cell or a cell-free system,relative to the amount or of unspliced XBP1 present in the cell orcell-free system. “The ratio of unspliced to spliced XBP1” refers to theamount of unspliced XBP1 compared to the amount of unspliced XBP1.“Increasing the ratio of spliced XBP1 to unspliced XBP1” encompassesincreasing the amount of spliced XBP1 or decreasing the amount ofunspliced XBP1 by, for example, promoting the degradation of unsplicedXBP1. Increasing the ratio of unspliced XBP1 to spliced XBP1 can beaccomplished, e.g., by decreasing the amount of spliced XBP1 or byincreasing the amount of unspliced XBP1. Levels of spliced and unsplicedXBP1 an be determined as described herein, e.g., by comparing amounts ofeach of the proteins which can be distinguished on the basis of theirmolecular weights or on the basis of their ability to be recognized byan antibody. In another embodiment described in more detail below, PCRcan be performed employing primers which span the splice junction toidentify unspliced XBP1 and spliced XBP1 and the ratio of these levelscan be readily calculated.

The present invention pertains to the novel discovery that XBP1 isconstitutively active in cancer-associated DCs, such as ovariancancer-asssociated DCs. In one embodiment, the present invention isdirected to the novel discovery that anti-tumor immunity is enhanced orincreased in a subject by directly or indirectly inhibiting XBP1. Inanother embodiment, the present invention is directed to the discoverythat ovarian cancer can be treated or reduced in a subject byadministering an effective amount of a direct or indirect inhibitor ofXBP1 to the subject.

IRE-1α

The term “IRE-1” or “IRE-1α” refers to an ER transmembraneendoribonuclease and kinase called “Serine/threonine-proteinkinase/endoribonuclease,” or alternatively, “Inositol-requiring protein1”, which oligomerizes and is activated by autophosphorylation uponsensing the presence of unfolded proteins, see, e.g., Shamu et al.,(1996) EMBO J. 15: 3028-3039. In Saccharomyces cerevisiae, the UPR iscontrolled by IREp. In the mammalian genome, there are two homologs ofIRE-1, IRE-1α and IRE-1β. IRE-1α is expressed in all cells and tissuewhereas IRE-1β is primarily expressed in intestinal tissue. Theendoribonucleases of either IRE-1α and IRE-1β are sufficient to activatethe UPR. Accordingly, as used herein, the term “IRE-1” includes, e.g.,IRE-1α, IRE-1β and IREp. In a preferred embodiment, IRE-1 refers toIRE-1α.

IRE-1 is a large protein having a transmembrane segment anchoring theprotein to the ER membrane. A segment of the IRE-1 protein has homologyto protein kinases and the C-terminal has some homology to RNAses.Over-expression of the IRE-1 gene leads to constitutive activation ofthe UPR. Phosphorylation of the IRE-1 protein occurs at specific serineor threonine residues in the protein.

IRE-1 senses the overabundance of unfolded proteins in the lumen of theER. The oligomerization of this kinase leads to the activation of aC-terminal endoribonuclease by trans-autophosphorylation of itscytoplasmic domains. IRE-1 uses its endoribonuclease activity to excisean intron from XBP1 mRNA. Cleavage and removal of a small intron isfollowed by re-ligation of the 5′ and 3′ fragments to produce aprocessed mRNA that is translated more efficiently and encodes a morestable protein (Calfon et al. (2002) Nature 415(3): 92-95). Thenucleotide specificity of the cleavage reaction for splicing XBP1 iswell documented and closely resembles that for IRE-p mediated cleavageof HAC1 mRNA (Yoshida et al. (2001) Cell 107:881-891). In particular,IRE-1 mediated cleavage of murine XBP1 cDNA occurs at nucleotides 506and 532 and results in the excision of a 26 base pair fragment. IRE-1mediated cleavage of XBP1 derived from other species, including humans,occurs at nucleotides corresponding to nucleotides 506 and 532 of murineXBP1 cDNA, for example, between nucleotides 502 and 503 and 528 and 529of human XBP1.

As used interchangeably herein, “IRE-1 activity,” “biological activityof IRE-1” or “functional activity IRE-1,” includes an activity exertedby IRE-1 on an IRE-1 responsive target or substrate, as determined invivo, or in vitro, according to standard techniques (Tirasophon et al.2000. Genes and Development Genes Dev. 2000 14: 2725-2736), IRE-1activity can be a direct activity, such as a phosphorylation of asubstrate (e.g., autokinase activity) or endoribonuclease activity on asubstrate e.g., XBP1 mRNA. In another embodiment, IRE-1 activity is anindirect activity, such as a downstream event brought about byinteraction of the IRE-1 protein with a IRE-1 target or substrate. AsIRE-1 is in a signal transduction pathway involving XBP1, modulation ofIRE-1 modulates a molecule in a signal transduction pathway involvingXBP1. Modulators which modulate an XBP1 biological activity indirectlymodulate expression and/or activity of a molecule in a signaltransduction pathway involving XBP1, e.g., IRE-1, PERK, eIF2α, or ATF6α.

The present invention provides the novel discovery that targeting theIRE-1α/XBP1 branch of the ER stress response in tumor-associated DCsinduces protective immune responses against cancer (e.g., ovariancancer). In some embodiment, the invention provides that inhibitingIRE-1α in tumor-associated DCs extends host survival by enhancinganti-tumor immunity.

Anti-Tumor Immunity

The immune system plays a critical role in protecting the host fromcancer. Notably, the tumor microenvironment is an important aspect ofcancer biology that contributes to tumor initiation, tumor progression,and responses to therapy. Cells and molecules of the immune system are afundamental component of the tumor microenvironment.

Since tumor tissue is characterized by a variety of antigens nottypically found in normal tissue, the immune system may mount aprotective response. In certain embodiments, these antigens are referredto as “tumor-associated antigens (TAAs).” Innate immunity against thetumor is invoked very quickly. Macrophages, which are programmed toattack and destroy tumor cells in the similar fashion that theyeliminate invading pathogens, are drawn to the tumor (Mantovani et al.,Nature Reviews Immunology 2011; 11, 519-531). With time, adaptiveanti-tumor immune responses develop. Dendritic cells migrate to thetumor as part of the innate immune response and serve as a link betweeninnate and adaptive immunity. In certain embodiments, dendritic cellsprocess tumor antigens and then directly interact with T and B cells,subsequently stimulating specific immune responses. The initial responseof the immune system to a tumor is to recruit lymphocytes in an attemptto clear the tumor. Tumor-infiltrating lymphocytes (TILs) includecytotoxic T lymphocytes (CTLs), helper T cells, and natural killer (NK)cells. Proteins associated with tumorigenesis or malignant growth mayalso stimulate humoral immunity (Suckow, The Veterinary Journal 2013;198, 28-33).

Coordination of the anti-tumor immune response requires communicationbetween cells of the immune system, mostly carried out by cytokines(Dranoff, Nature Reviews Cancer 2004; 4, 11-22). For example,interleukin (IL)-6 produced by T lymphocytes and macrophages enhancesthe proliferation of both T and B lymphocytes. Likewise,interferon-gamma (IFNγ) is produced by NK cells, T lymphocytes,macrophages, and B lymphocytes and enhances tumor antigen presentationalong with cell-mediated cytotoxicity.

The predominant cell type within tumor stroma is the fibroblast.Cancer-associated fibroblasts produce a variety of factors that promoteproliferation and progression of cancer, including osteonectin, vascularendothelial growth factor (VEGF), and matrix metalloproteinases (MMPs).Many of these factors are widely produced by normal cells and thereforethe immune system restricts itself from attacking these targets (Rasanenand Vaheri, Experimental Cell Research 2010; 316, 2713-2722).

In certain embodiments, the presence of a tumor serves as evidence thatcancerous cells have successfully avoided immune elimination. This mayoccur due to immune selection pressure that favors growth of tumors thatare less immunogenic (Dunn et. al, Nature Immunology 2002; 3, 991-998).Tumors may also escape through the expression of anti-apoptoticmolecules (Reed, Current Opinion in Oncology 1999; 11, 68-75). Factorssuch as VEGF, soluble Fas, and transforming growth factor (TGF)-β, whichare produced by tumor cells and tumor stroma, can suppress anti-tumorimmune response (Ben-Baruch, Seminars in Cancer Biology 2006; 16, 38-52;Whiteside, Seminars in Cancer Biology 2006; 16, 3-15). Tumor stromalcells create an environment in which cancerous cells are exposed togrowth factors while avoiding immune recognition. For example,thrombospondin-1 is produced by stromal cells and leads to immunesuppression via activation of TGF-3 (Silze et. al, International Journalof Cancer 2004; 108, 173-180).

Harnessing the inherent ability of T cells to eliminate tumor cellsrepresents the most promising anti-cancer strategy since the developmentof chemotherapy, as demonstrated most recently by the dramatic shrinkageof melanoma in response to checkpoint blockers anti-CTLA4 and anti-PD 1.In addition, recent reports demonstrate that adoptively transferredanti-tumor T cells (expanded from resected tumor specimens orgenetically manipulated) can elicit robust and long-lasting anti-tumorresponses (Bollard et al., 2007; Dudley et al., 2002; Leen et al., 2006;Morgan et al., 2006). However, in most cases, the optimal cytotoxicactivity of such tumor-reactive T cells is drastically reduced becausecancer-associated DCs are unable to support T cell function. (Barnett etal., 2005; Conejo-Garcia et al., 2004; Cubillos-Ruiz et al., 2009;Curiel et al., 2003; Huarte et al., 2008). The present invention revealsfor the first time that DC-specific deletion of XBP1 can extend hostsurvival by converting immunosuppressive tDCs into potent activators ofType 1 immunity in ovarian cancer-infiltrating T cells. Indeed,therapeutic silencing of XBP1 in tDCs using siRNA-encapsulatingnanocarriers reversed their immunosuppressive phenotype andsignificantly prolonged host survival by inducing protective anti-tumorimmune responses. Novel and more effective therapeutic strategies areneeded to improve the dismal prognosis of metastatic ovarian cancerpatients. The present invention demonstrates for the first time thefeasibility and significant immunotherapeutic potential of targeting ERstress-driven XBP1 in tDCs using a safe and effectivenanotechnology-based system that may slow or prevent the usuallyinevitable recurrence observed in metastatic ovarian cancer patients whohave been “optimally” debulked. The present invention also provides thattargeting the aberrant ER stress response in innate immune cells of thetumor microenvironment may also represent a viable therapeutic strategyto confront other lethal cancers that normally co-opt the immuneresponse to promote malignant progression. Hence, the present inventionprovides for the first time that targeting the IRE-1α/XBP1 arm of the ERstress response in cancer-bearing hosts could be used to inhibit tumorgrowth while simultaneously inducing robust anti-tumor immunity.

In one embodiment, the present invention is directed to the noveldiscovery that constitutive activation of XBP1 in tumor-associateddendritic cells allows tumors to manipulate DC function and elude immunecontrol. In another embodiment, the present invention is directed to thediscovery that ablaiting XBP1 expression in tumor-associated dendriticcells enhances or induces anti-tumor immunity and extends host survival.In certain embodiments, inhibition of XBP1 enhances infiltration ofactivated T cells at tumor locations. In certain embodiments, inhibitionof XBP1 enhances the capacity of infiltrating T cells to respond totumor antigens.

Dendritic Cells

Dendritic cells (DC) are key regulators of both innate and adaptiveimmunity, and the array of immunoregulatory functions exhibited by thesecells is dictated by their differentiation, maturation, and activationstatus. A major role of these cells is the induction of immunity topathogens; however, recent data demonstrates that DCs are also criticalregulators of anti-tumor immune responses. In certain embodiments, thegeneration of protective anti-tumor immunity depends on the presentationof tumor antigens by DCs to T cells. The control of DC survival plays animportant role in regulating T cell activation and function.

DCs initiate an immune response by presenting a captured antigen, in theform of peptide-major histocompatability complex (MHC) moleculecomplexes, to naïve T cells in lymphoid tissues. Upon interaction withDCs, naïve CD4+ and CD8+ T cells can differentiate into antigen-specificeffector cells. DCs also play a direct role in humoral immunity byinteracting with B cells and indirectly by inducing the expansion anddifferentiation of CD4+ helper T cells.

Presentation of antigens to mount an immune response is one of theprimary functions of dendritic cells. Tumor-associated DCs have the samefunction but the antigens are typically tumor-associated (TAA). Tumorscan prevent antigen presentation and the establishment of tumor-specificimmune responses through a variety of mechanisms. For example, tumorsswitch the differentiation of monocytes into macrophages and not DCs, orprevent the priming of tumor-specific T cells by DCs, through themediation of IL-2 and macrophage colony-stimulating factor. In certainembodiments, tumors can interfere with DC maturation. For example, tumorcells can secrete IL-10 which leads to antigen-specific anergy of DCs.In certain embodiments, DCs are tumor-associated DCs (tDCs). These tDCsare present in the tumor microenvironment. The present invention,provides the novel discovery that tumors rely on DC-intrinsic XBP1 toelude immune control. The present invention also provides the noveldiscovery that inhibiting XBP1 in tumor-associated DCs extends hostsurvival by enhancing anti-tumor immunity.

In one embodiment, the present invention is directed to the noveldiscovery that the ER stress sensor XBP1 is constitutively active incancer-associated DCs, such as ovarian cancer-associated DCs. In anotherembodiment, the present invention pertains to the discovery that thelipid peroxidation byproduct 4-HNE triggers ER stress and XBP1activation in DCs. In another embodiment, the present invention pertainsto the discovery that XBP1 regulates lipid metabolism and antigenpresentation by tumor-associated DCs.

XBP1 and Dendritic Cells

XBP1 is a transcription factor expressed in dendritic cells andactivated by IRE-1α, an ER transmembrane kinase and endoribonuclease.XBP1 functions to regulate the ER stress response by maintaining ERhomeostasis and preventing activation of cell death pathways caused bysustained ER stress. The ER stress response, or unfolded proteinresponse (UPR) is activated when unfolded proteins accumulate in the ERand functions to regulate the balance between homeostasis and apoptosis.In certain embodiments, XBP1 plays a role in DC differentiation andsurvival. For example, XBP1-deficient cells are more sensitive toapoptosis (Iwakoshi 2007).

The instant inventors have discovered that XBP1 is constitutively activein tumor-associated dendritic cells.

In one embodiment, the present invention provides the novel discoverythat XBP1 is a driver of dendritic cell dysfunction in the tumormicroenvironment. In another embodiment, the present invention providesthat XBP1 is constitutively active in cancer-associated dendritic cells,such as ovarian cancer-associated dendritic cells. In certainembodiments, XBP1-deficient tDCs demonstrate enhanced antigen-presentingcapacity. In certain embodiments, silencing or inhibiting XBP1 improveshost survival and induces anti-tumor immunity. In certain embodiments,the induction of anti-tumor immunity is carried out by activatedinfiltrating T cells that respond to tumor antigens.

XBP1 and Ovarian Cancer

During tumor development and progression, cancer cells encountercytotoxic conditions such as hypoxia, nutrient deprivation, and low pHdue to inadequate vascularization (Hanahan, D., et al. 2011. Cell 144,646-74). To maintain survival and growth in the face of thesephysiologic stressors, a set of adaptive response pathways are induced.One adaptive pathway well studied in other contexts is the unfoldedprotein response (UPR), which is induced by factors affecting theendoplasmic reticulum (ER) such as changes in glycosylation, redoxstatus, glucose availability, calcium homeostasis or the accumulation ofunfolded or misfolded proteins (Hetz, C, et al. 2011. Physiol Rev 91,1219-43). Notably, features of the tumor microenvironment, such ashypoxia and nutrient deprivation, can disrupt ER homeostasis by theperturbation of aerobic processes such as oligosaccharide modification,disulphide bond formation, isomerization, and protein quality controland export (Wouters, B. G., et al. 2008. Nat Rev Cancer 8, 851-64). Inmammalian cells, the UPR is mediated by three ER-localized transmembraneprotein sensors: Inositol-requiring transmembrane kinase/endonuclease-1(IRE-1), PKR-like ER kinase (PERK) and activating transcription factor 6(ATF6) (Walter, P., et al. 2011. Science 334, 1081-6). Of these, IRE-1is the most evolutionarily conserved branch. An increase in the load offolding proteins in the ER activates IRE-1, an ER-resident kinase andendoribonuclease that acts as an ER-stress sensor. Activated IRE-1removes a 26 bp intron from XBP1 mRNA and results in a frame shift inthe coding sequence, with the spliced form encoding a 226 amino acidtranscriptional activation domain (Calfon, M., et al. 2002. Nature 415,92-6; Yoshida, H., et al. 2001. Cell 107, 881-91). In contrast to theunspliced XBP1 (XBP1u), which is unstable and quickly degraded, splicedXBP1 (XBP1s) is stable and is a potent inducer of target genes thatorchestrate the cellular response to ER stress (Hetz, C, et al. 2011.Physiol Rev 91, 1219-43).

As described in detail above, the UPR is a major cellular stressresponse pathway activated in tumors that allows them to adapt to thestresses of the tumor microenvironment. Several studies have reported onthe activation of the UPR in various human tumors and its relevance tocombinatorial therapy (Carrasco, D. R., et al. 2007. Cancer Cell 11,349-36; De Raedt, T., et al. 2011. Cancer Cell 20, 400-413; Healy, S.J., et al. 2009. Eur J Pharmacol 625, 234-246; Ma, Y., et al. 2004. NatRev Cancer 4, 966-977; Mahoney, D. J., et al. 2011. Cancer Cell 20,443-456). However, the role of the UPR and XBP1 in anti-tumor immunityis largely unknown. Here, the instant inventors have identified apreviously unknown function of XBP1 in anti-tumor immunity and ovariancancer. Here, it is demonstrated that constitutive activation of XBP1, akey component of the most evolutionarily conserved branch of the UPR,allows tumors to evade immune control by crippling normal DC function.Furthermore, XBP1 deletion and/or silencing inhibits tumor growth and/orprogression and induces robust anti-tumor immunity.

Tumors progress when the host fails to provide an effective anti-tumorimmune response. Prior to the discovery of the present invention, therole of XBP1 in anti-tumor immunity was unknown. Herein the instantinventors have identified a role for XBP1 in eluding immune control andtherefore inducing the progression of tumor development, such as ovariantumor development. In certain embodiments, the present inventionprovides that XBP1 is constitutively active in tumor-associateddendritic cells present in ovarian tumors compared to normal dendriticcells. In certain embodiments, the present invention provides thatconstitutive activation of XBP1 is critical for the initiation and rapidprogression of ovarian tumors. Notably, XBP1 deficient tDCs fail toinitiate or progress tumor development (e.g., ovarian tumors). Incertain embodiments, the rapid progression of ovarian tumors is a resultof constitutively active XBP1 in tumor-associated dendritic cells. Incertain embodiments, the present invention provides that tumorprogression and/or tumor burden is reduced in XBP1-deficient tDCs,indicating an important role for XBP1 in the development and progressionof ovarian tumors.

Lipid Metabolism and Peroxidation

Cancer-associated DCs accumulate substantial amounts of oxidized lipids,and this abnormal process negatively regulates their antigen-presentingcapacity (Herber et al., 2010; Ramakrishnan et al., 2014). As providedby the instant invention, transcriptional and functional analyses oftDCs devoid of XBP1 suggest that this conserved ER stress sensorfacilitates the aberrant lipid accumulation commonly observed indysfunctional cancer-associated DCs.

Lipid oxidation by reactive oxygen species (ROS) generates reactivebyproducts such as the unsaturated aldehyde 4-hydroxy-trans-2-nonenal(4-HNE), which has been shown to induce protein-folding stress byforming stable adducts with ER-resident chaperones (Vladykovskaya etal., 2012). For the first time, the present invention provides that4-HNE, a lipid peroxidation byproduct available in the human and mouseovarian cancer microenvironment, can rapidly trigger robust ER stressand XBP1 activation in naïve DCs. Interestingly, 4-HNE has beendemonstrated to induce the unfolded protein response in endothelialcells by forming covalent adducts with ER resident chaperones, a processthat promotes vascular inflammation (Vladykovskaya et al., 2012). The ERstress response has been linked to lipid biosynthesis (Lee et al., 2008;Sriburi et al., 2004). However, the present invention indicates thatenhanced intracellular lipid accumulation by tDCs required ROSgeneration and IRE-1α/XBP1 activation. Interestingly, exposure toXBP1-activating 4-HNE has been shown to promote fat accumulation inworms and mice (Singh et al., 2008; Singh et al., 2009). Thus, in someembodiments, the present invention provides that reactive metabolicbyproducts in the tumor microenvironment, like 4-HNE, can perpetuate ERstress in infiltrating immune cells such as tDCs. Constitutiveactivation of the IRE-1α/XBP1 arm through this process subsequentlypromotes abnormal intracellular lipid accumulation in tDCs viaupregulation of lipid biosynthetic genes, which ultimately inhibitstheir natural capacity to support T cell-mediated anti-tumor responses.

In one embodiment, the present invention provides that XBP1-deficienttDCs display marked downregulation of genes involved in lipid metabolicpathways. These lipid biosynthetic genes are rapidly upregulated innaïve DCs exposed to 4-HNE, an XBP1-activation lipid peroxidationbyproduct. This aberrant lipid accumulation by tDCs obstructs theirnormal antigen processing and presentation capacity (Herber et al.,2010; Ramakrisnan et al., 2014).

In one embodiment, the present invention provides that the lipidperoxidation byproduct 4-HNE triggers ER stress and XBP1 activation inDCs. In another embodiment, the present invention provides that XBP1regulates lipid metabolism and antigen presentation by tumor-associatedDCs. In one embodiment, the present invention provides that XBP1deficient tDCs have reduced intracellular lipid accumulation andimproved antigen presenting capacity, leading to enhanced intra-tumoralT cell activation and increased host survival.

Therapeutic Targeting of the UPR

In the present invention, an unexpected role for the ER stress sensorXBP1 as a central driver of DC malfunction in the tumor microenvironmenthas been discovered. These findings unveil a new mechanistic paradigmwhereby a lethal cancer exploits the most conserved arm of the ER stressresponse in tumor-resident DCs to disrupt their lipid homeostasis, altertheir local antigen-presenting capacity and ultimately evade Tcell-mediated immune control. While the ER stress response, andespecially XBP1 activation, has been previously shown to operate incancer cells to promote tumorigenesis, the present invention now revealsthat this integrated cellular pathway further supports malignantprogression by inhibiting the development of protective anti-tumorimmunity via manipulation of normal DC function.

The upstream kinase and endoribonuclease IRE-1, which drives thesplicing of XBP1 mRNA, is a viable drug target. Recently, two groupshave identified specific IRE-1 endoribonuclease inhibitors (Papandreou,I., et al. 2011. Blood 117, 1311-1314; Volkmann, K., et al. 2011. J BiolChem 286, 12743-12755). Intriguingly, these compounds efficientlyinhibit XBP1 splicing in vivo and dramatically impair tumor growth in axenograft model (Mahoney, D. J., et al. 2011. Cancer Cell 20, 443-456;Papandreou, I., et al. 2011. Blood 117, 1311-1314; Volkmann, K., et al.2011. J Biol Chem 286, 12743-12755). While large-scale small moleculescreens have provided potentially promising candidates that target theIRE-1/XBP1 pathway, attention needs to be paid to the specificity andcytotoxity of these compounds in vivo. Recent advances in solving thecrystal structure of IRE-1 (Korennykh, A. V., et al. 2009. Nature 457,687-693; Lee, K. P., et al. 2008. Cell 132, 89-100; Zhou, J., et al.2006. Proc Natl Acad Sci USA 103, 14343-14348) should accelerate thedesign of more potent and specific IRE-1 inhibitors. The use of UPRinhibitors in combination with standard chemotherapy may greatly enhancethe effectiveness of anti-tumor therapies.

The methods of the invention using inhibitory compounds which inhibitthe expression, processing, post-translational modification, or activityof XBP1 or a molecule in a biological pathway involving XBP1, such asIRE-1α, can be used to induce anti-tumor immunity or in the treatment ofcancer (e.g., ovarian cancer). In one embodiment of the invention, aninhibitory compound can be used to inhibit (e.g., specifically inhibit)the expression, processing, post-translational modification, or activityof spliced XBP1. In another embodiment, an inhibitory compound can beused to inhibit (e.g., specifically inhibit) the expression, processing,post-translational modification, or activity of unspliced XBP1.

Inhibitory compounds of the invention can be, for example, intracellularbinding molecules that act to directly or indirectly inhibit theexpression, processing, post-translational modification, or activity ofXBP1 or a molecule in a biological pathway involving XBP1, for example,IRE-1α. As used herein, the term “intracellular binding molecule” isintended to include molecules that act intracellularly to inhibit theprocessing expression or activity of a protein by binding to the proteinor to a nucleic acid (e.g., an mRNA molecule) that encodes the protein.Examples of intracellular binding molecules, described in further detailbelow, include antisense nucleic acids, intracellular antibodies,peptidic compounds that inhibit the interaction of XBP1 or a molecule ina biological pathway involving XBP1 (e.g., IRE-1α) and a target moleculeand chemical agents that specifically or directly inhibit XBP1 activityor the activity of a molecule in a biological pathway involving XBP1(e.g., IRE-1α).

In one embodiment, an inhibitory compound of the invention is anantisense nucleic acid molecule that is complementary to a gene encodingXBP1 or a molecule in a signal transduction pathway involving XBP1,(e.g., IRE-1α or a molecule with which XBP1 interacts), or to a portionof said gene, or a recombinant expression vector encoding said antisensenucleic acid molecule. The use of antisense nucleic acids todownregulate the expression of a particular protein in a cell is wellknown in the art (see e.g., Weintraub, H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996) N. Eng. J. Med.334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation92:1981-1993; Mercola, D. and Cohen, J. S. (1995) Cancer Gene Ther.2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R. W.(1994) Nature 372:333-335). An antisense nucleic acid molecule comprisesa nucleotide sequence that is complementary to the coding strand ofanother nucleic acid molecule (e.g., an mRNA sequence) and accordinglyis capable of hydrogen bonding to the coding strand of the other nucleicacid molecule. Antisense sequences complementary to a sequence of anmRNA can be complementary to a sequence found in the coding region ofthe mRNA, the 5′ or 3′ untranslated region of the mRNA or a regionbridging the coding region and an untranslated region (e.g., at thejunction of the 5′ untranslated region and the coding region).Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid is designed so as to be complementary to a regionpreceding or spanning the initiation codon on the coding strand or inthe 3′ untranslated region of an mRNA. Given the known nucleotidesequence for the coding strand of the XBP1 gene and thus the knownsequence of the XBP1 mRNA, antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick base pairing. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of an XBP1. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. Similarly, antisense nucleic acidstargeting IRE-1α can also be designed according to the rules of Watsonand Crick base pairing. An antisense nucleic acid of the invention canbe constructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. To inhibit expression in cells, oneor more antisense oligonucleotides can be used.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which all or a portion of a cDNA hasbeen subcloned in an antisense orientation (i.e., nucleic acidtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest). The antisenseexpression vector can be in the form of, for example, a recombinantplasmid, phagemid or attenuated virus. The antisense expression vectorcan be introduced into cells using a standard transfection technique.

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a protein tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. An example of a route ofadministration of an antisense nucleic acid molecule of the inventionincludes direct injection at a tissue site (e.g. a tumor site).Alternatively, an antisense nucleic acid molecule can be modified totarget selected cells (for example, tumor-associated dendritic cells)and then administered systemically. For example, for systemicadministration, an antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein.

In yet another embodiment, an antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An a-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In certain embodiments, an antisense nucleic acid molecule of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave mRNA transcripts to thereby inhibit translationmRNAs. Alternatively, gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a gene(e.g., an XBP1 promoter and/or enhancer) to form triple helicalstructures that prevent transcription of a gene in target cells. Seegenerally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene,C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)Bioassays 14(12):807-15.

In another embodiment, a compound that promotes RNAi can be used toinhibit expression of XBP1 or a molecule in a biological pathwayinvolving XBP1. The term “RNA interference” or “RNAi”, as used herein,refers generally to a sequence-specific or selective process by which atarget molecule (e.g., a target gene, protein or RNA) is downregulated.In certain embodiments, the process of “RNA interference” or “RNAi”features degradation of RNA molecules, e.g., RNA molecules within acell, said degradation being triggered by an RNA agent. Degradation iscatalyzed by an enzymatic, RNA-induced silencing complex (RISC). RNAioccurs in cells naturally to remove foreign RNAs (e.g., viral RNAs).Natural RNAi proceeds via fragments cleaved from free dsRNA which directthe degradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes. RNA interference (RNAi) is apost-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287,2431-2432 (2000); Zamore, P. D., et al. Cell 101, 25-33 (2000). Tuschl,T. et al. Genes Dev. 13, 3191-3197 (1999); Cottrell T R, and Doering TL. 2003. Trends Microbiol. 11:37-43; Bushman F. 2003. Mol Therapy.7:9-10; McManus M T and Sharp P A. 2002. Nat Rev Genet. 3:737-47). Theprocess occurs when an endogenous ribonuclease cleaves the longer dsRNAinto shorter, e.g., 21-23-nucleotide-long RNAs, termed small interferingRNAs or siRNAs. As used herein, the term “small interfering RNA”(“siRNA”) (also referred to in the art as “short interfering RNAs”)refers to an RNA agent, preferably a double-stranded agent, of about10-50 nucleotides in length (the term “nucleotides” including nucleotideanalogs), preferably between about 15-25 nucleotides in length, morepreferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength, the strands optionally having overhanging ends comprising, forexample 1, 2 or 3 overhanging nucleotides (or nucleotide analogs), whichis capable of directing or mediating RNA interference.Naturally-occurring siRNAs are generated from longer dsRNA molecules(e.g., >25 nucleotides in length) by a cell's RNAi machinery (e.g.,Dicer or a homolog thereof). The smaller RNA segments then mediate thedegradation of the target mRNA. Nanoparticle-encapsulated siRNA can alsobe used to downregulate a target molecule. Kits for synthesis of RNAiare commercially available from, e.g. New England Biolabs or Ambion. Inone embodiment one or more of the chemistries described above for use inantisense RNA can be employed in molecules that mediate RNAi.

Alternatively, a compound that promotes RNAi can be expressed in a cell,e.g., a cell in a subject, to inhibit expression of XBP1 or a moleculein a biological pathway involving XBP1, such as IRE-1α. In contrast tosiRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) andenter at the top of the gene silencing pathway. For this reason, shRNAsare believed to mediate gene silencing more efficiently by being fedthrough the entire natural gene silencing pathway. The term “shRNA”, asused herein, refers to an RNA agent having a stem-loop structure,comprising a first and second region of complementary sequence, thedegree of complementarity and orientation of the regions beingsufficient such that base pairing occurs between the regions, the firstand second regions being joined by a loop region, the loop resultingfrom a lack of base pairing between nucleotides (or nucleotide analogs)within the loop region. shRNAs may be substrates for the enzyme Dicer,and the products of Dicer cleavage may participate in RNAi. shRNAs maybe derived from transcription of an endogenous gene encoding a shRNA, ormay be derived from transcription of an exogenous gene introduced into acell or organism on a vector, e.g., a plasmid vector or a viral vector.An exogenous gene encoding an shRNA can additionally be introduced intoa cell or organism using other methods known in the art, e.g.,lipofection, nucleofection, etc.

The requisite elements of a shRNA molecule include a first portion and asecond portion, having sufficient complementarity to anneal or hybridizeto form a duplex or double-stranded stem portion. The two portions neednot be fully or perfectly complementary. The first and second “stem”portions are connected by a portion having a sequence that hasinsufficient sequence complementarity to anneal or hybridize to otherportions of the shRNA. This latter portion is referred to as a “loop”portion in the shRNA molecule. The shRNA molecules are processed togenerate siRNAs. shRNAs can also include one or more bulges, i.e., extranucleotides that create a small nucleotide “loop” in a portion of thestem, for example a one-, two- or three-nucleotide loop. The stemportions can be the same length, or one portion can include an overhangof, for example, 1-5 nucleotides.

In certain embodiments, shRNAs of the invention include the sequences ofa desired siRNA molecule described supra. In such embodiments, shRNAprecursors include in the duplex stem the 21-23 or so nucleotidesequences of the siRNA, desired to be produced in vivo.

Another type of inhibitory compound that can be used to inhibit theexpression and/or activity of XBP1 or a molecule in a biological pathwayinvolving XBP1 (for example, IRE-1α) is an intracellular antibodyspecific for said protein. The use of intracellular antibodies toinhibit protein function in a cell is known in the art (see e.g.,Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al.(1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBS Letters274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen,S-Y. et al. (1994) Human Gene Therapy 5:595-601; Duan, L et al. (1994)Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994) Proc.Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol.Chem. 269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys.Res. Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell.

In another embodiment, an inhibitory compound of the invention is apeptidic compound derived from the XBP1 amino acid sequence or the aminoacid sequence of a molecule in a biologicalon pathway involving XBP1.

The peptidic compounds of the invention can be made intracellularly incells by introducing into the cells an expression vector encoding thepeptide. Such expression vectors can be made by standard techniquesusing oligonucleotides that encode the amino acid sequence of thepeptidic compound. The peptide can be expressed in intracellularly as afusion with another protein or peptide (e.g., a GST fusion). Alternativeto recombinant synthesis of the peptides in the cells, the peptides canbe made by chemical synthesis using standard peptide synthesistechniques. Synthesized peptides can then be introduced into cells by avariety of means known in the art for introducing peptides into cells(e.g., liposome and the like).

In addition, dominant negative proteins (e.g., of XBP1 or IRE-1α) can bemade which include XBP1 or IRE-1α (e.g., portions or variants thereof)that compete with native (i.e., wild-type) molecules, but which do nothave the same biological activity. Such molecules effectively decrease,e.g., XBP1 IRE-1α activity in a cell.

Other inhibitory agents that can be used to specifically inhibit theactivity of XBP1 or a molecule in a biological pathway involving XBP1(e.g., IRE-1α) are chemical compounds that directly inhibit expression,processing, post-translational modification, and/or activity of XBP1.Such compounds can be identified using screening assays that select forsuch compounds, as described in detail above as well as using other artrecognized techniques.

In certain embodiments, targeting XBP1 inhibits tumor growth. In certainembodiments, targeting XBP1 inhibits ovarian tumor growth.

The present invention provides that constitutive activation of the UPRin dendritic cells prevents a proper anti-tumor immune response. Incertain embodiments, targeting XBP1 results in an anti-tumor immuneresponse. In certain embodiments, the anti-tumor immune response is theproliferation and infiltration of T cells targeted for a specifictumor-associated antigen.

Screening Assays

In one aspect, the invention features methods for identifying compoundsuseful in enhancing or inducing anti-tumor immunity in a subject, suchcompounds have the potential for therapeutic use in the treatment ofcancer, such as ovarian cancer. In other aspects, the invention featuresmethods for identifying compounds useful in inhibiting the growth ofovarian cancer cells, such compounds having potential therapeutic use inthe treatment of ovarian cancer. As described herein, the instantinvention is based, at least in part, on the discovery of a previouslyunknown role for XBP1 in anti-tumor immunity and ovarian cancer, such arole being linked to anti-tumor immunity directed by tumor-associateddendritic cells. In exemplary aspects the invention features methods foridentifying compounds useful for inducing or enhancing anti-tumorimmunity and inhibiting the growth of ovarian cancer cells, the methodsfeaturing screening or assaying for compounds that modulate, e.g.,activate or increase, or inhibit or decrease, the activation ofIRE-1/XBP1. In exemplary aspects, the methods comprise: providing anindicator composition comprising XBP1, or biologically active portionsthereof; contacting the indicator composition with each member of alibrary of test compounds; selecting from the library of test compoundsa compound of interest that interacts with XBP1, or biologically activeportions thereof; and contacting ovarian cancer cells with the compoundof interest, wherein the ability of the compound to enhance or induceanti-tumor immunity in the subject is indicated by the ability of thecompound to inhibit growth of ovarian cancer cells as compared to thegrowth of ovarian cancer cells in the absence of the compound.

In another exemplary aspect, the methods comprise: providing anindicator composition comprising XBP1, or biologically active portionsthereof; contacting the indicator composition with each member of alibrary of test compounds; and selecting from the library of testcompounds a compound of interest that decreases the activity of XBP1, orbiologically active portions thereof, wherein the ability of a compoundto induce anti-tumor immunity or inhibit growth of ovarian cancer cellsis indicated by a decrease in the activation as compared to the amountof activation in the absence of the compound.

As used herein, the term “contacting” (i.e., contacting a cell e.g. acell, with a compound) includes incubating the compound and the celltogether in vitro (e.g., adding the compound to cells in culture) aswell as administering the compound to a subject such that the compoundand cells of the subject are contacted in vivo. The term “contacting”does not include exposure of cells to an XBP1 modulator that may occurnaturally in a subject (i.e., exposure that may occur as a result of anatural physiological process).

As used herein, the term “test compound” refers to a compound that hasnot previously been identified as, or recognized to be, a modulator ofthe activity being tested. The term “library of test compounds” refersto a panel comprising a multiplicity of test compounds.

As used herein, the term “indicator composition” refers to a compositionthat includes a protein of interest (e.g., XBP1 or a molecule in abiological pathway involving XBP1, such as IRE-1α), for example, a cellthat naturally expresses the protein, a cell that has been engineered toexpress the protein by introducing one or more of expression vectorsencoding the protein(s) into the cell, or a cell free composition thatcontains the protein(s) (e.g., purified naturally-occurring protein orrecombinantly-engineered protein(s)).

As used herein, the term “cell” includes prokaryotic and eukaryoticcells. In one embodiment, a cell of the invention is a bacterial cell.In another embodiment, a cell of the invention is a fungal cell, such asa yeast cell. In another embodiment, a cell of the invention is avertebrate cell, e.g., an avian or mammalian cell. In a preferredembodiment, a cell of the invention is a murine or human cell. As usedherein, the term “engineered” (as in an engineered cell) refers to acell into which a nucleic acid molecule e.g., encoding an XBP1 protein(e.g., a spliced and/or unspliced form of XBP1) has been introduced.

As used herein, the term “cell free composition” refers to an isolatedcomposition, which does not contain intact cells. Examples of cell freecompositions include cell extracts and compositions containing isolatedproteins.

The cells used in the instant assays can be eukaryotic or prokaryotic inorigin. For example, in one embodiment, the cell is a bacterial cell. Inanother embodiment, the cell is a fungal cell, e.g., a yeast cell. Inanother embodiment, the cell is a vertebrate cell, e.g., an avian or amammalian cell. In a preferred embodiment, the cell is a human cell. Thecells of the invention can express endogenous XBP1 or can be engineeredto do so. For example, a cell that has been engineered to express theXBP1 protein can be produced by introducing into the cell an expressionvector encoding the protein. Recombinant expression vectors can be usedfor expression of XBP1.

In another embodiment, the indicator composition is a cell freecomposition. XBP1 expressed by recombinant methods in a host cells orculture medium can be isolated from the host cells, or cell culturemedium using standard methods for protein purification. For example,ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies can be used to produce a purified or semi-purified proteinthat can be used in a cell free composition. Alternatively, a lysate oran extract of cells expressing the protein of interest can be preparedfor use as cell-free composition.

Pharmaceutical Compositions and Modes of Administration

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. Agents include,but are not limited to, in vitro synthetically prepared chemicalcompositions, antibodies, antigen-binding regions, and combinations andconjugates thereof. In certain embodiments, an agent can act as anagonist, antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for pharmaceuticalcomnpositions comprising an XBP1 inhibitor or an inhibitor of a moleculein a biological pathway involving XBP1 (e.g., IRE-1α) with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. In certain embodiments, acceptableformulation materials preferably are nontoxic to recipients at thedosages and concentrations employed. In certain embodiments, theformulation material(s) are for s.c. and/or I.V. administration. Incertain embodiments, the pharmaceutical composition can containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolality, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In certain embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-IHCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyciodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or inmunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chiorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In certain embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceuticalcomposition will be determined by one skilled in the art depending upon,for example, the intended route of administration, delivery format anddesired dosage. See, for example, Remington's Pharmaceutical Sciences,supra. In certain embodiments, such compositions may influence thephysical state, stability, rate of in vivo release and rate of in vivoclearance of an XBP1 inhibitor or an inhibitor of a molecule in abiological pathway involving XBP1 (e.g., IRE-1α).

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In certainembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can furtherinclude sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising an XBP1 inhibitor or an inhibitorof a molecule in a biological pathway involving XBP1 (e.g., IRE-1α) canbe prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising an XBP1 inhibitor or an inhibitor of a molecule in abiological pathway involving XBP1 (e.g., IRE-1α) can be formulated as alyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired XBP1inhibitor or an inhibitor of a molecule in a biological pathwayinvolving XBP1 (e.g., IRE-1α) in a pharmaceutically acceptable vehicle.In certain embodiments, a vehicle for parenteral injection is steriledistilled water in which an XBP1 inhibitor or an inhibitor of a moleculein a biological pathway involving XBP1 (e.g., IRE-1α) is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, an XBP1 inhibitor or aninhibitor of a molecule in a biological pathway involving XBP1 (e.g.,IRE-1α) can be formulated as a dry powder for inhalation. In certainembodiments, an inhalation solution comprising an XBP1 inhibitor or aninhibitor of a molecule in a biological pathway involving XBP1 (e.g.,IRE-1α) can be formulated with a propellant for aerosol delivery. Incertain embodiments, solutions can be nebulized. Pulmonaryadministration is further described in PCT application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, an XBP1 inhibitor or aninhibitor of a molecule in a biological pathway involving XBP1 (e.g.,IRE-1α) that is administered in this fashion can be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. In certain embodiments, acapsule can be designed to release the active portion of the formulationat the point in the gastrointestinal tract when bioavailability ismaximized and pre-systemic degradation is minimized. In certainembodiments, at least one additional agent can be included to facilitateabsorption of an XBP1 inhibitor or absorption of an inhibitor of amolecule in a biological pathway involving XBP1 (e.g., IRE-1α). Incertain embodiments, diluents, flavorings, low melting point waxes,vegetable oils. lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of an XBP1 inhibitor or an inhibitor of a molecule ina biological pathway involving XBP1 (e.g., IRE-1α) in a mixture withnon-toxic excipients, which are suitable for the manufacture of tablets.In certain embodiments, by dissolving the tablets in sterile water, oranother appropriate vehicle, solutions can be prepared in unit-doseform. In certain embodiments, suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving an XBP1 inhibitor or aninhibitor of a molecule in a biological pathway involving XBP1 (e.g.,IRE-1α) in sustained- or controlled-delivery formulations. In certainembodiments, techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829, which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et aL, Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); E1⁾ 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcornposition comprising an XBP1 inhibitor or an inhibitor of a moleculein a biological pathway involving XBP1 (e.g., IRE-1α), to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which an XBP1 inhibitor or an inhibitor ofa molecule in a biological pathway involving XBP1 (e.g., IRE-1α) isbeing used, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an XBP1 inhibitor or an inhibitor of amolecule in a biological pathway involving XBP1 (e.g., IRE-1α) in theformulation used. In certain embodiments, a clinician will administerthe composition until a dosage is reached that achieves the desiredeffect. In certain embodiments, the composition can therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infu sion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. In certain embodiments, appropropriate dosages can beascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,systemically, locally, through injection by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intratumoral, intramuscular, subcutaneously,intra-ocular, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. In certainembodiments, the compositions can be administered by bolus injection orcontinuously by infusion, or by implantation device. In certainembodiments, individual elements of the combination therapy may beadministered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In certain embodiments, it can be desirable to use apharmaceutical composition comprising an XBP1 inhibitor or an inhibitorof a molecule in a biological pathway involving XBP1 (e.g., IRE-1α) inan ex vivo manner. In such instances, cells, tissues and/or organs thathave been removed from the patient are exposed to a pharmaceuticalcomposition comprising an XBP1 inhibitor or an inhibitor of a moleculein a biological pathway involving XBP1 (e.g., IRE-1α), after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

In certain embodiments, an XBP1 inhibitor or an inhibitor of a moleculein a biological pathway involving XBP1 (e.g., IRE-1α) can be deliveredby implanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides. In certain embodiments, such cells can be animal or humancells, and can be autologous, heterologous, or xenogeneic. In certainembodiments, the cells can be immortalized. In certain embodiments, inorder to decrease the chance of an immunological response, the cells canbe encapsulated to avoid infiltration of surrounding tissues. In certainembodiments, the encapsulation materials are typically biocompatible,semipermeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

In other embodiments, ex vivo treatment of dendritic cells with aninhibitor of XBP1 (e.g., siRNA) can be used to activate, induce, enhanceor promote the antigen presenting capacity of dendritic cells. Theactivated dendritic cells can then be administered to a subject. In someembodiments, the dendritic cells may be tumor-associated dendriticcells.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures and the sequence listing, are herebyincorporated by reference.

EXAMPLES Experimental Procedures Tissues, Mice and Cell Lines

Stage III-IV human ovarian carcinoma specimens and malignant ascitessamples were procured through Surgical Pathology at Weill CornellMedical College/NewYork-Presbyterian Hospital under an approved protocolwhere research samples remained totally unidentified. Tumor single cellsuspensions were generated as previously described (Conejo-Garcia etal., 2005). Malignant peritoneal ascites samples from patients withmetastatic ovarian cancer were centrifuged for 10 min at 1300 rpm andred blood cells were lysed prior to FACS analysis. Mice were housed atthe animal facilities of Harvard School of Public School, Weill CornellMedical College, or The Wistar Institute. The Institutional Animal Careand Use Committee approved all animal experiments described in thisstudy. XBP1^(f/f) mice were generated as previously described (Lee etal., 2008) and have been backcrossed at least 15 generations ontoC57BL/6 mice. Wild type, OT-1 transgenic, Itgax-Cre (CD11c-Cre) andRag2-deficient mice, all in a full C57BL/6 background, were purchasedfrom Jackson Laboratories (Bar Harbor, Me.).

Double transgenic LSL-K-ras^(G12D/+)p53^(loxp/loxp) (p53/K-ras) micewere generated by obtaining LSL-K-ras^(tm4Tyj) (Jackson et al., 2001)and Trp53^(tmlBrn) (Jonkers et al., 2001) from the NCI mouse models ofhuman cancer consortium and bred to a full C57BL/6 background aspreviously reported (Scarlett et al., 2012). p53/K-ras mice wereirradiated two consecutive days with 650 rads followed by reconstitutionwith bone marrow from XBP1^(f/f) or XBP1^(f/f) CD11c-Cre mice. 8 weekspost bone-marrow reconstitution autochthonous ovarian tumors wereinitiated by delivery of adenovirus-expressing Cre recombinase (ADV-Cre)into the ovarian bursa as previously reported (Dinulescu et al., 2005;Flesken-Nikitin et al., 2003; Scarlett et al., 2012). Seven weeks aftertumor initiation, mice were sacrificed. Tumors, approximately 2-3 cm indiameter, were resected under sterile conditions after euthanizing themouse. Specimens were then minced into pieces <3 mm in diameter anddigested for 1 hour at 37° C. in RPMI containing 2 mg/mL collagenaseType D and 1 mg/ml DNAse I. The digested tissue pieces were then pressedthrough a 70 μm strainer to create a single cell suspension. Red bloodcells were lysed using ACK lysis buffer and pellets from single-cellsuspensions were resuspended to 50-100×10⁶ cells/ml in freezing media(FBS containing 10% DMSO) and incubated on ice for 30 minutes. Tubeswere then transferred to −80° C. for long-term storage.

Parental ID8 or aggressive ID8-Defb29/Vegf-A intraperitoneal ovariantumors were generated as previously described (Conejo-Garcia et al.,2004; Roby et al., 2000). Briefly, 1-2×10⁶ tumor cells were injectedinto wild type C57BL/6 mice or conditional XBP1-deficient mice.Implanted animals progressively developed multiple peritoneal masses andeventually massive ascites in −35 days (ID8-Defb29/Vegf-A) or in ˜2months (parental ID8). Mice were weighted weekly to monitor malignantascites accumulation and animals with severe abdominal distension werehumanely euthanized.

Isolation of Human and Mouse DCs

Human patient ovarian cancer-associated DCs (CD45⁺CD3⁻CD20⁻CD11c⁺DEC205⁺) were sorted from tumor single cell suspensions ormalignant ascites using flow cytometry, following the gating strategydescribed in FIG. 1. During sorting, viable cells were identified usingthe LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Life Technologies).Mouse ovarian cancer-associated DCs (CD45⁺CD11c⁺CD11b⁺MHC-II⁺CD8α⁻) weresorted from single-cell suspensions of p53/K-ras-driven ovarian tumorsor from peritoneal wash (10 ml 1×PBS) or total malignant ascites samplesfrom mice bearing aggressive ID8-Defb29/Vegf-A intraperitoneal ovariancancer, following the gating strategy described in FIG. 1. Allfluorescently-labeled antibodies were from BioLegend. Control sDCs(CD45⁺CD11c⁺CD11b⁺MHC-II⁺CD8⁺) were FACS sorted from spleens of naïve orovarian cancer-bearing mice using Collagenase D and DNAse I treatmentfollowed by incubation with the indicated antibodies.

Reagents and In Vitro Cellular Treatments

Murine recombinant cytokines were purchased from Peprotech. Cobaltchloride, Tunicamycin (used at 1 μg/ml), Tiron (used at 100-500 μM) andVitamin E (α-tocopherol, used at 50-100 μM) were from Sigma. DCFDAstaining was utilized for intracellular ROS detection (Abcam). Purified4-HNE was obtained from Cayman Chemical and 4-HNE-protein adducts inascites samples and DCs were detected and quantified through competitiveELISA (Cell Biolabs). TOFA (Cayman Chemical) was used at a finalconcentration of 5 μg/ml to inhibit fatty acid synthesis in DCs byblocking the synthesis of malonyl-CoA by acetyl-CoA carboxylase. TheIRE-1α-specific inhibitor 4μ8c (Millipore) was used at a finalconcentration of 10 μM.

Conventional and Quantitative RT-PCR

Total RNA from human samples was isolated using the miRVANA miRNAisolation Kit (Life Technologies). RNA from mouse samples was isolatedusing the Qiazol reagent (Qiagen). 0.1-1 tg of RNA were used to generatecDNA using the High Capacity cDNA Reverse Transcription Kit (LifeTechnologies). Human and mouse Xbp1 splicing assays were performed asdescribed (Lee et al., 2003a; Martinon et al., 2010) using conventionalReverse Transcription PCR (RT-PCR) and primers shown in Table 1. Geneexpression analysis was done via Reverse Transcription quantitative PCR(RT-qPCR) using a Stratagene Mx3005 instrument and SYBR green I (LifeTechnologies). Murine XBP1s transcript expression was determined using aprobe that spans the spliced-out version as previously demonstrated(Reimold et al., 2001). All primers used in this study are described inTable 1.

Western Blot

5×10⁶ sDC or tDC were washed twice in 1× cold PBS and nuclear proteinswere purified using the Nuclear Extraction Kit (Life Technologies).Proteins were quantified using the BCA method (Pierce) and 15-20 μg ofnuclear proteins were separated via SDS-PAGE and transferred ontonitrocellulose membranes following standard procedures. Anti-mouse XBP1s(GL Biochem) was raised in rabbit using a peptide corresponding to theXBP1s C-terminus, and was used at a 1:500 dilution for immunoblotting.Goat anti-mouse Lamin B (Santa Cruz) was used at 1:2000. HRP-conjugatedsecondary antibodies to rabbit and mouse (Santa Cruz) were used at a1:2000 dilution. SuperSignal West Femto (Pirce) was used asChemiluminescent Substrate and blots were imaged using a FluorChemEinstrument (ProteinSimple).

RNA-Seq and DC Transcriptional Profile

tDCs were sorted from peritoneal wash samples of XBP1^(f/f) orXBP1^(f/f) CD11c-Cre female mice (n=3/group) bearing aggressiveID8-Defb29/Vegf-A ovarian tumors for 3 weeks. Total RNA was isolatedusing the miRVANA miRNA isolation Kit (Life Technologies) and furtherconcentrated via RNeasy MinElute columns (Qiagen). RNA quality andintegrity was confirmed in an Agilent Bioanalyzer 2100. In all casesRINs were 9.50 or higher. mRNA libraries were generated and sequenced atthe Epigenomics Facility of Weill Cornell Medical College. Readsproduced from 5 lbp single end sequencing run were aligned against mousegenome (mm9) using Bowtie v0.12.8 (Langmead et al., 2009) algorithm.Mouse mm9 transcriptome information was obtained from UCSC GenomeBrowser and RSEM algorithm (Li and Dewey, 2011) was used to calculatenumber of aligned tags for each gene. Differential expression betweentwo groups were tested by EdgeR (Robinson and Oshlack, 2010) andsignificance was defined using a False Discovery Rate (FDR) cutoff of0.15. The most up-to-date gene information (official symbol anddescription) was obtained from NCBI Entrez information on May 15, 2014.Normalized expression RPKM values (Reads Per Kilobase of transcript perMillion mapped reads) values were generated by EdgeR and used todemonstrate gene expression across samples as color-coded fold change ofexpression in a sample versus average expression across all samples.Functional enrichment analysis was done using QIAGEN's Ingenuity PathwayAnalysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). For IPAupstream analysis, only regulators significant at p<10⁻⁶ with predictedactivation/inhibition states (Z-score>2) were considered. Significantlyaffected biological processes were tested by using NCBI DAVID (Huang daet al., 2009) software using level 3 of GO biological processes andconsidering only FDR<0.15 results that showed at least 20 genes thatconstitute at least ⅔ of all involved in the process genes specificallyup or down-regulated.

Flow Cytometry and Lipid Staining

Intracellular lipid content in DCs was evaluated via flow cytometryusing 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene(BODIPY 493/503, Life Technologies) as previously reported (Herber etal., 2010). Briefly, 5×10⁶ cells from total spleen single cellsuspensions or malignant peritoneal wash samples were conventionallystained for surface markers using fluorescently-labeled antibodies thatdo not overlap with BODIPY 493/503, namely CD11c-APC, CD45-APC-Cy7 andCD11b-Pacific Blue. Cells were washed twice with 1×PBS and stained with500 μl of BODIPY 493/503 at 0.5 mg/ml in PBS for 15 min at roomtemperature in the dark. Cells were washed twice and analyzed by flowcytometry. BODIPY 493/503 staining was detected in the PE channel. Forintracellular cytokine staining, 5×10⁶ cells isolated from malignantperitoneal wash samples of ovarian cancer-bearing mice were stimulatedfor 6h in 10% FBS complete RPMI containing PMA (Calbiochem), lonomycin(Calbiochem) and Brefeldin A (BioLegend). Cells were collected andstained for surface markers and intracellular cytokines following theFoxP3/Transcription Factor Staining buffer set (eBioscience). Allantibodies were from BioLegend. Flow cytometry was performed on a LSRIIinstrument (BD Biosciences). Cell populations were sorted fromperitoneal washes (10 ml 1×PBS) of ovarian carcinoma-bearing mice orfrom human ascites or tumor single-cell suspensions using a FACSAriasorter (BD Biosciences). Flow cytometry data was analyzed using FlowJoversion 9 or 10.

Transmission Electron Microscopy and Lipidomics

tDC were sorted from the peritoneal cavity of XBP1^(f/f) or XBP^(f/f)CD11c-Cre female mice bearing ID8-Defb29/Vegf-A ovarian tumors for 3 to4 weeks as shown in FIG. 1. Cells were washed twice with 1×PBS andpellets were fixed and sectioned for electron microscopy analysis(performed at the Electron Microscopy and Histology Core Facility ofWeill Cornell Medical College) following standard methods.Alternatively, cell pellets from 0.5-1×10⁶ sorted tDCs were frozen andtotal intracellular lipids were extracted and quantitatively analyzedvia LC-MS at the Lipidomics Core Facility of Wayne State UniversitySchool of Medicine.

Antigen Processing and Presentation

For in vitro antigen presentation experiments, tDC or sDC were sortedfrom the peritoneal cavity or spleen of XBP1^(f/f) or XBP1^(f/f)CD11c-Cre female mice bearing ID8-Defb29/Vegf-A ovarian tumors for 4weeks (FIG. 1). tDCs were pulsed for overnight 50 μg/ml of full-lengthendotoxin-free OVA (SIGMA, Grade VII) in the presence or absence ofVitamin E (50 μg/ml) in media containing 25% cell-free ovarian cancerascites supernatants. DCs were washed twice and cocultured for 3 dayswith CFSE-labeled CD8⁺ T cells immunopurified from OT-1 mice at a 1:10(DC to T cell) ratio, as previously described (Scarlett et al., 2009).

For in vivo antigen presentation experiments, wild type C57BL/6 femalemice bearing ID8-Defb29/Vegf-A ovarian tumors for three weeks wereintraperitoneally injected with 0.6 mg of full length endotoxin-free OVA(SIGMA, grade VII) and 3 hours later, mice were left untreated orinjected with siRNA-PEI nanoparticles (see below). 18 hours later, micewere transferred intraperitoneally with 2×10⁶ CFSE-labeled T cellsnegatively purified from OT-1 transgenic mice. Peritoneal wash samples(10 mL) were collected after 72 hours and analyzed for CFSE dilution viaFACS. Data were analyzed using FlowJo version 10. Division Indexdetermines the average number of cell divisions that a cell in theoriginal population has undergone. Proliferation Index shows the totalnumber of divisions of only responding (proliferating) cells.Replication Index is the fold-expansion of the responding cells, thusindicating the expansion capability of the replicating cells.

Preparation of siRNA-PEI Nanoparticles and Therapeutic In Vivo Silencing

Endotoxin-free rhodamine-labeled and unconjugated polyethylenimine (PEI)for in vivo experiments “in vivo-jetPEI” was purchased from PolyPlusTransfection. To generate siRNA-PEI nanocomplexes, 50 μg of siRNA werecomplexed with “in vivo-jetPEI” at N/P ratio of 6, following therecommendations of the manufacturer and previously optimized conditions(Cubillos-Ruiz et al., 2009). All siRNA oligonucleotides were from IDTand included 2′-OMe modified nucleotides and specific phosphorothioatelinkages, as previously reported (Piret et al., 2002). Sequences for thesense and antisense strands are as follows: siLuc sense:5′-CuUACgcUGAguaCUUcGAdTsdT-3′, siLuc antisense:5′-UCgAAGUACUCAGCgUAAGdTdsT-3′, siXBP1 sense:5′-cAcccuGAAuucAuuGucudTsdT-3′, siXBP1 antisense:5′-AGAcAAUGAAUUcAGGGUGdTsdT-3′. silREla sense:5′-AuGccGAAGuucAGAuGGAdTsdT-3′, silREla antisense:5′-UCcAUCUGAACUUCGGcAUdTsdT-3′. 2′-OMe modified nucleotides are in lowercase. Phosphorothioate linkages are represented by “s” and “d” indicatesDNA bases.

For in vivo biodistribution, phenotypic and silencing experiments, micebearing ID8-Defb29/Vegf-A tumors for 3-4 weeks were intraperitoneallyinjected with rhodamine-labeled siXBP1-PEI or siLuc-PEI nanoparticles(50 tg of siRNA complexed with rhodamine-labeled “in vivo-jetPEI” at N/P6, per mouse). Rhodamine⁺CD45⁺CD11c⁺CD11b⁺MHC-II⁺ tDC were sorted after3 days for downstream molecular biology analysis. For repeated siRNAtreatments, wild-type C57BL/6 female mice were intraperitoneallyinjected with 1×10⁶ aggressive ID8-Defb29/Vegf-A ovarian carcinomacells, and mice received nanocomplexes (50 μg of siRNA complexed with“in vivo-jetPEI” at N/P 6, per mouse) at days 12, 16, 20, 24, 28 and 32after tumor implantation.

Anti-Tumor Immune Responses and ELISA

Mice were intraperitoneally injected with ID8-Defb29/Vegf-A ovariancancer cells and treated with siRNA-PEI nanoparticles (n=3/group) atdays 8, 13, 18, and 23 after challenge. Total splenic T cells orFicoll-enriched leukocytes (2-3×10⁵) from peritoneal wash samples wereobtained 4 days after the last treatment (day 27) and cocultured in 10%FBS RPMI with 2-3×10⁴ bone marrow-derived DCs previously pulsedovernight with irradiated ID8-Defb29/Vegf-A ovarian cancer cells.Supernatants were collected after 48-72 h of stimulation. IFN-γ andGranzyme B secretion cells was determined by ELISA using theReady-SET-Go Kit (eBioscience).

Statistical Analysis

Unless noted otherwise, all experiments were repeated at least two timesand results were similar between repeats. The correlation between CHOPexpression in tDC and human intra-tumoral T cell infiltration wasanalyzed using the Spearman's Rank coefficient. Animal experiments usedbetween 3 and 6 mice per group. A P value <0.05 was considered to bestatistically significant. All statistical analyses were done usingGraph Pad Prism 5.0. Differences between the means of experimentalgroups were calculated using a two-tailed unpaired Student's t test.Error bars represent standard error of the mean from independent samplesassayed within the represented experiments. Survival rates were comparedusing the Log-Rank test. All survival experiments used at least 6mice/group. This number provides a 5% significance level and 95% powerto detect differences in survival of 20% or greater.

Example 1: Constitutive XBP1 Activation in Ovarian Cancer-Associated DCs

Innate myeloid cells with phenotypic and functional attributes ofregulatory DCs commonly infiltrate ovarian tumors (Conejo-Garcia et al.,2004; Huarte et al., 2008; Scarlett et al., 2012). Rather than inducinganti-cancer immunity, these dysfunctional DCs facilitate malignantprogression by preventing the activation and expansion of tumor-reactiveT cells (Cubillos-Ruiz et al., 2010). To analyze XBP1 activation inhuman and mouse ovarian cancer-associated DCs, CD45⁺CD3⁻CD20⁻CD11⁺DEC205⁺ tDCs were isolated via FACS from human patient ovariantumors or metastatic ovarian cancer ascites samples. MurineCD45⁺CD11c⁺MHC-II⁺ CD11b⁺CD8α⁻ tDCs were isolated from advancedp53/K-ras-driven ovarian tumors or from malignant ascites of micebearing aggressive ID8-Defb29/Vegf-A ovarian carcinoma for 4-5 weeks andtheir identity as bonafide classical DCs was confirmed by quantifyingClec9A/DNGR-1 and Zbtb46 expression (FIG. 1). Splicing of the Xbp1 mRNAwas evaluated using conventional PCR.

Malignant peritoneal fluid samples from patients with metastatic ovariancancer were centrifuged for 10 min at 1300 rpm and total cells were usedfor FACS analysis. Solid primary or metastatic ovarian tumors weremechanically dissociated as described above. In both cases,CD45⁺CD3⁻CD20⁻CD11c⁺DEC205⁺ tDCs were sorted via FACS for RNA extractionand gene expression quantification via RT-qPCR. The percentage ofCD45⁺CD3⁺ T cells present in each sample was correlated with CHOP mRNAexpression levels in sorted tDCs from the same specimen (FIG. 2F).

Notably, tumor-associated DCs (tDCs) isolated from multiple humanpatient ovarian cancer specimens (FIGS. 1A and 1B) or from preclinicalmodels of aggressive primary and metastatic ovarian cancer (FIGS. 1C-1E)(Conejo-Garcia et al., 2004; Scarlett et al., 2012) exhibitedconstitutive splicing of the Xbp1 mRNA (FIG. 2A), a molecular eventessential for generating fully functional XBP1 (Yoshida et al., 2001).

Expression of the indicated transcripts was determined by RT-qPCR (FIG.2B, 2D, 2E) (data are normalized to endogenous levels of Actb in eachsample. CD45⁺CD11c⁺MHC-II⁺CD11b⁺CD8⁻ sDCs were isolated from spleens ofnaïve or tumor-bearing mice), and quantitative analyses demonstratedincreased expression of total and spliced Xbp1 mRNA in tDCs, comparedwith closely related CD8α− splenic DCs (sDCs) (FIG. 1E) isolated eitherfrom naïve or ovarian cancer-bearing mice (FIG. 2B).

Western blot analysis was used to assay the spliced form of XBP1 (XBP1s)protein expression in nuclear extracts obtained from the indicated DCs.Consistently, tDCs exhibited augmented XBP1 protein levels in thenucleus compared with control DCs obtained from non-tumor sites (FIG.2C). Further confirming these findings, RT-PCR analysis showed markedupregulation of canonical XBP1 target genes ERdj4 and Sec61a1(Acosta-Alvear et al., 2007; Lee et al., 2003b) (FIG. 2D), as well asincreased expression of general ER stress response markers Hspa5 (BiP)and Ddit3 (CHOP) was evidenced only in tDCs (FIG. 2E).

The expression of CHOP in tDCs sorted from human patient ovarian cancerspecimens was determined using RT-qPCR. Interestingly, CHOP expressionlevels in tDCs negatively correlated with T cell infiltration in severalhuman ovarian cancer specimens analyzed, suggesting a potential role forER-stressed tDCs in regulating anti-tumor immune responses (FIG. 2F).Together, these data indicate that DCs in the ovarian cancermicroenvironment exhibit severe ER stress and robust XBP1 activation.

Example 2: Byproducts of Lipid Peroxidation Trigger ER Stress in DCs

One goal of the present invention was to investigate how the tumormicroenvironment influences the functional status of XBP1 in DCs. Inparticular, one aim of the present invention was to investigate whethercancer-derived factors could participate in triggering ER stress andXBP1 activation in tumor-infiltrating DCs, and determine whether thisprocess could impact the detrimental function of these innate immunecells in hosts with ovarian cancer. Interestingly, neithertumorigenic/immunosuppressive cytokines commonly enriched at tumor sitesnor hypoxia-mimicking conditions caused robust XBP1 activation in DCs(FIG. 3). In particular, naïve sDCs were isolated and stimulated for 24hwith cytokines (e.g., IL-10, TGF-β, VEGFα, KC, CCL3, IL-6) atconcentrations ranging from 5 ng/ml to 25 ng/ml (FIG. 3A-D). Cells werealso exposed for 24 hours to increasing concentrations of cobaltchloride (CoCl₂), a chemical inducer of the HIF1α pathway that mimicslow oxygen conditions (Piret et al. 2002) (FIG. 3E-F). Splicing andupregulation of XBP1 was determined by RT-qPCR analysis (FIG. 3).

Recent reports demonstrate that abnormal intracellular accumulation ofperoxided lipids is a common feature of dysfunctional DCs infiltratingmultiple human and mouse cancers (Herber et al., 2010; Ramakrishnan etal., 2014). Importantly, lipid oxidation by reactive oxygen species(ROS) generates reactive byproducts such as the unsaturated aldehyde4-hydroxy-trans-2-nonenal (4-HNE), which has been shown to induceprotein-folding stress by forming stable adducts with ER-residentchaperones (Vladykovskaya et al., 2012). Whether these reactive aldehydebyproducts could trigger ER stress in DCs was investigated. Ovariancancer-associated DCs demonstrated significantly higher amounts ofintracellular lipids and augmented ROS levels in comparison with controlnon-malignant sDCs isolated from the same host or from naïve mice (FIGS.4A and 4B). Consistent with active lipid peroxidation taking place attumor sites, cell-free ovarian cancer ascites of human and mouse originexhibited high levels of 4-HNE-protein adducts (FIG. 4C). Accordingly,intracellular 4-HNE-protein adducts were also readily found in tDCsisolated from these malignant samples (FIG. 4D). 4-HNE generation inmouse tDCs exposed to cell-free ovarian cancer ascites decreased uponexposure to the common antioxidant Vitamin E (FIG. 4E). Incubation ofnaïve sDCs with increasing concentrations of purified 4-HNE efficientlyformed covalent adducts with intracellular proteins (FIG. 4F) andrapidly triggered splicing of the Xbp1 mRNA in an IRE-1-dependent manner(FIGS. 4G and 4H). Consistently, upregulation of the canonicalXBP1-dependent, ER-resident chaperone ERdj4 (FIG. 4I) as well as robustinduction of the general ER stress response markers BiP and CHOP (FIG.4J) was rapidly evidenced in 4-HNE treated DCs. These data demonstratethat 4-HNE, a lipid peroxidation byproduct readily available in theovarian cancer microenvironment, triggers strong ER stress and XBP1activation in DCs.

Example 3: DC-Intrinsic XBP1 is Necessary for Optimal Ovarian CancerProgression

To determine how sustained XBP1 activity in tDCs might influencemalignant progression, aggressive orthotopic ovarian tumors inconditional knockout female mice lacking functional XBP1 in DCs weredeveloped (FIG. 5). To this end, mice whose exon 2 of Xbp1 is flanked bytwo loxP sites (Lee et al., 2008) were crossed with mice expressing Crerecombinase under control of the integrin alpha X (Itgax) promoter(hereafter referred to as CD11c-Cre) (FIG. 5). In this system,Cre-mediated recombination is predominant in conventional DCs, while lowamounts of recombination are detected in lymphocytes, NK cells and othermyeloid cells (Caton et al., 2007).

tDCs were sorted from the peritoneal cavity of mice bearing metastaticID8-VegfA-Def29b ovarian tumors for 4-5 weeks as described in FIG. 1.Total CD11c⁺ DCs were magnetically immunopurified from spleens (sDC) orfrom GMCSF-polarized bone marrow cultures obtained from XBP1^(f/f) (wildtype) or XBP1^(f/f)CD11c-Cre (conditional knockout) mice. Total splenicT cells and CD11b⁺F4/80⁺ macrophages (M0) were also isolated as controlpopulations. Deletion efficiency was determined by RT-qPCR using primersthat selectively amplify exon 2 of Xbp1 (see methods) (FIG. 5B). sDCsfrom naïve XBP1^(f/f) or XBP1^(f/f) CD11c-Cre mice were left untreatedor stimulated for 12 h with the ER stressor Tunicamycin at 1 μg/ml.Induction of canonical XBP1 target genes ERdj4 and Sec61 uponstimulation was determined via RT-qPCR. In all cases, data werenormalized to endogenous Actb expression in each sample. Data arerepresentative of at least three independent experiments with similarresults (FIG. 5C).

It had been previously been reported that overall DC survival wascompromised upon extensive ablation of XBP1 in early hematopoieticprecursors (Iwakoshi et al., 2007). In contrast, conditional deletion ofXBP1 through CD11c-controlled Cre expression solely affected theproportion and number of splenic CD8α⁺ DCs (FIG. 6). Other immune cellpopulations in the spleen remained unaffected (FIG. 6) and the frequencyof tumor-infiltrating DCs, which are mainly CD8α⁻ (FIGS. 1 and 6), wasnot altered in conditional knockout mice bearing orthotopic ovariancancers (FIG. 6). These data suggest that XBP1 is dispensable for theoptimal survival of DCs in the ovarian cancer microenvironment.Strikingly, the development and metastatic capacity of p53/K-ras-drivenprimary ovarian tumors (Scarlett et al., 2012) was profoundlycompromised in irradiated hosts reconstituted with bone marrow fromXBP1-deficient (XBP1^(f/f) CD11c-Cre) donors, compared with controlhosts transplanted with bone marrow from XBP1-sufficient (XBP1^(f/f))littermates (FIGS. 7A-7C). These data demonstrate that XBP1 expressionin CD11c⁺ DCs is crucial for the initiation and rapid progression ofovarian tumors.

Since the vast majority of ovarian cancers are diagnosed at advancedstages, when the disease has spread throughout the peritoneal cavity,the next goal was to define how DC-derived XBP1 impacts the progressionof orthotropic tumors that closely recapitulate the physiopathology ofhuman metastatic ovarian cancer (Conejo-Garcia et al., 2004). Notably,ovarian cancer-bearing female mice lacking functional XBP1 in DCsdemonstrated reduced peritoneal tumor burden (FIGS. 7D and 7E), impairedascites accumulation (FIG. 7F), and diminished tumor-inducedsplenomegaly (FIG. 7G) compared with control gene-sufficient(XBP1^(f/f)) littermates. Consequently, tumor-bearing mice deficient forXBP1 in DCs showed a marked increase in survival compared with controllittermates (FIG. 7H). Similar survival results were observed inXBP1^(f/f) CD11c-Cre mice developing parental orthotopic ovarian tumors(Roby et al., 2000) that do not ectopically express Defb29 and Vegf-A(FIG. 7I). Together, these results demonstrate for the first time thatXBP1 expression in DCs is necessary for the aggressive and acceleratedprogression of primary and metastatic ovarian cancers in preclinicalmodels of disease.

Example 4: XBP1 Activation Disrupts Lipid Homeostasis in DCs

To elucidate how XBP1 confers pro-tumorigenic activity in ovariancancer-associated DCs, a comparison of the transcriptional profile ofwild type vs. XBP1-deficient DCs residing in malignant ovarian cancerascites was performed. 416 genes that were significantly downregulatedwere identified, while 237 genes showed significantly higher expressiondue to XBP1 deficiency (Table 2). Significantly altered gene subsetswere analyzed to identify transcriptional regulators that may explainthe observed mRNA changes. Several predicted affected regulators wereidentified (FIG. 8A) with the expected XBP1 as a top hit. Additionally,XBP1 was a key node in the top gene network found from the data (FIG.8B). Confirming sustained ER stress in DCs infiltrating ovarian cancer,multiple direct XBP1 target genes and genes implicated in the unfoldedprotein response (Acosta-Alvear et al., 2007; Lee et al., 2003b) weremarkedly repressed in XBP1-deficient tDCs (FIG. 9A). Expression of knownRegulated IRE-1α-dependent Decay (RIDD) target mRNAs (Hetz et al., 2013;Hollien et al., 2009) was indistinguishable between wild type andXBP1-deficient tDCs (FIG. 8C), indicating that IRE-1α is notartificially overactivated in this cell type due to the absence offunctional XBP1 (So et al., 2012). A search was then performed for anybiological processes affected by XBP1-deficiency and 4 significantlyenriched functional categories were found (FIG. 8D). Of particularinterest, tDCs devoid of XBP1 displayed marked downregulation ofmultiple genes involved in lipid metabolic pathways (FIG. 9B). In lightof the strong association between enhanced lipid accumulation and DCdysfunction in cancer (Herber et al., 2010; Ramakrishnan et al., 2014),the role of XBP1 as a potential mediator of this process wasinvestigated. Consistent with sustained ER stress at tumor locations,ovarian cancer-associated DCs exhibited severe upregulation of multipleXBP1-controlled lipid metabolism genes (FIG. 9B), including Agpat6, Fasnand Lpar1, compared with control DCs in lymphoid tissue (FIG. 10A). Ofnote, these lipid biosynthetic genes were rapidly upregulated in naïvesDCs exposed to the XBP1-activating lipid peroxidation byproduct 4-HNE(FIG. 10B). Most importantly, ovarian cancer-associated DCs lacking XBP1showed reduced intracellular lipid content compared with their wild typecounterparts (FIG. 9C). Further supporting these findings,XBP1-deficient tDC demonstrated a marked decrease in the number ofcytosolic lipid droplets (FIGS. 9D and 9E) as well as reducedintracellular levels of total triglycerides (FIG. 9F) compared withXBP1-sufficient tDC. Other lipid classes remained unaffected in tDCslacking XBP1 (FIG. 10C) and these observations were confirmed byanalyzing cell-free ascites supernatants (FIG. 10D). Decreasedintracellular lipid content in XBP1-deficient tDCs did not occur due todefective expression of genes encoding scavenger receptors implicated inextracellular lipid uptake, including Cd36, Cd68 and Msr1 (FIG. 10E).Notably, exposure to cell-free ovarian cancer ascites augmented theintracellular lipid content of tDCs and this process that was preventedby treatment with TOFA, an inhibitor of fatty acid synthesis blockingthe synthesis of malonyl-CoA by acetyl-CoA carboxylase (FIGS. 9G and10F). Pharmacological inhibition of IRE-1α/XBP1 activation using 4μ8calso restricted the observed ascites-induced lipid biogenesis in tDCs(FIGS. 9G and 10F). In addition, consistent with the function of ROS askey generator of XBP1-activating 4-HNE (FIG. 4), reduced intracellularlipid content was also evidenced in tDCs treated with the global ROSscavenger Vitamin E, but not with the superoxide-specific scavengerTiron (FIGS. 9G and 10F). Taken together, the transcriptional andfunctional data indicate that sustained activation of ER stress sensorXBP1 disrupts intracellular lipid homeostasis in ovariancancer-associated DCs.

Example 5: XBP1-Deficient tDCs Support T Cell Activation

Aberrant lipid accumulation by cancer-associated DCs has beendemonstrated to obstruct their normal antigen processing andpresentation capacity (Herber et al., 2010; Ramakrishnan et al., 2014).Whether XBP1-deficient tDCs with reduced levels of intracellular lipidsmight support rather than repress T cell activation and function attumor sites was investigated. While surface expression of MHC moleculesand CD80 remained unaltered, tDCs devoid of XBP1 demonstrated higherlevels of costimulatory receptors CD40, CD83 and CD86 (FIGS. 11A and11B). XBP1-deficiency did not alter the antigen-presenting capacity ofcontrol CD8α⁻ sDCs (FIG. 12). However, XBP1-deficient tDCs pulsed withfull-length OVA in the presence of cell-free malignant ascites moreefficiently induced the expansion of OT-1 T cells compared with wildtype tDCs (FIGS. 11C and 11D). Consistent with impaired lipidaccumulation upon global ROS scavenging and IRE-1α/XBP1 inhibition (FIG.9G), pre-treatment of regulatory wild type tDC with Vitamin Ephenocopied the enhanced antigen-presenting capacity exhibited byXBP1-deficient tDCs in this in vitro assay (FIGS. 11C and 11D). Furthersupporting these findings, conditional knockout mice developingaggressive orthotopic ovarian tumors demonstrated a marked increase inthe proportion of infiltrating CD44⁺IFNγ-secreting CD8⁺ and CD4⁺ T cellsat tumor sites compared with XBP1-sufficient control littermates (FIGS.11E and 11F). Together, these data indicate that constitutive XBP1activation promotes intracellular lipid accumulation in and immunetolerance by ovarian cancer-associated DCs.

Example 6: Therapeutic XBP1 Silencing in tDCs Extends Host Survival byInducing Anti-Tumor Immunity

T cells are the only immune population known to exert significantpressure against ovarian cancer progression (Callahan et al., 2008;Curiel et al., 2003; Hamanishi et al., 2007; Han et al., 2008; Sato etal., 2005; Zhang et al., 2003). Indeed, it has been demonstrated thatthe magnitude of intra-tumoral T cell infiltration strongly correlateswith a better outcome in ovarian cancer patients (Zhang et al., 2003).To investigate whether targeting XBP1 function in vivo and in situ couldbe used to restore the immunogenic attributes of ovariancancer-associated DCs and hence, promote the function of anti-tumor Tcells, polyethylenimine (PEI)-based nanoparticles encapsulating siRNAwere utilized (Cubillos-Ruiz et al., 2012; Cubillos-Ruiz et al., 2009).Wild type C57BL/6 female mice were injected with 1-2×10̂6ID8-Defb29/Vegf-A ovarian cancer cells and mice received a singleinjection of control (luciferase-specific) or XBP1-targeting siRNAencapsulated in rhodamine-labeled PEI-based nanocomplexes 3-4 weekslater (FIG. 13). These nanocomplexes are preferentially and avidlyengulfed by abundant phagocytic tDCs upon intraperitoneal (i.p.)injection, a process that enables selective in vivo gene silencing inthis leukocyte subset (Cubillos-Ruiz et al., 2009). Importantly,PEI-based nanoparticles inherently activate ovarian cancer-associatedDCs by triggering TLR signaling and therefore exhibit potentimmunoadjuvant activity against tumors (Cubillos-Ruiz et al., 2009).Confirming our previous findings (Cubillos-Ruiz et al., 2012;Cubillos-Ruiz et al., 2009), nanoparticles were selectively taken-up bytDCs present in malignant ascites of mice bearing metastatic ovariancancer (FIG. 13A). PEI-based nanocomplexes delivering Xbp1-specificsiRNA induced 50-60% gene silencing in target tDC compared with controlnanoparticles encapsulating luciferase-targeting siRNA (FIG. 13B). Thefunctional effects of gene silencing were confirmed by the decreasedexpression of the canonical XBP1 target ERdj4, as well as lipidbiosynthesis-related Fasn and Scd2 in tDCs engulfing Xbp1-specificnanocomplexes (FIG. 13C). Due to selective tDC targeting in vivo, Xbp1mRNA levels remained unaltered in non-DC leukocytes or cancer cells ofthe tumor microenvironment after nanoparticle injection (not shown).Supporting the findings using tDC from conditional XBP1 knockout mice,the in vivo proliferation of adoptively transferred CFSE-labeled OT-1 Tcells in the ovarian cancer microenvironment was significantly enhancedin mice pulsed i.p. with full-length OVA when XBP1 expression wasspecifically silenced in tDCs (FIGS. 14A-14C). Therapeuticadministration of siRNA-PEI nanocomplexes selectively targetingtDC-derived XBP1 further reduced the total number of metastatic cancercells in the peritoneal cavity (FIG. 14D) and consequently diminishedthe accumulation of malignant ascites (FIG. 14E). Importantly, theseeffects occurred concomitantly with the enhanced infiltration ofendogenous antigen-experienced/activated T cells at tumor locationscompared with treatments using control nanoparticles (FIG. 14F). Indeed,silencing XBP1 expression in ovarian cancer-associated DCs markedlyenhanced the capacity of infiltrating T cells to respond to tumorantigens, as evidenced by ex vivo IFN-γ and Granzyme B secretion assaysusing bone marrow-derived DCs pulsed with tumor antigens (FIG. 14G).These data reinforce the concept that abrogating XBP1 function in tDCsdrives the activation and expansion of endogenous anti-tumor T cells attumor sites. Splenic T cells isolated from mice treated withXBP1-silencing nanocomplexes also demonstrated improved responses uponexposure to tumor antigens in similar re-call assays (FIG. 14H),indicating development of enhanced anti-tumor memory responses aftertargeting XBP1 in tDCs. Together, these results demonstrate thattherapeutic silencing of XBP1 in ovarian cancer-associated DCs can boostendogenous anti-tumor immune responses in vivo.

To determine whether selective abrogation of the IRE-1α/XBP1 pathway intDCs had significant therapeutic effects, mice bearing aggressiveorthotopic ovarian tumors were i.p. treated with saline, non-targetingor gene-specific siRNA-PEI nanocomplexes. Treatments started 12 daysafter tumor implantation and injections were administered every 4 daysfor a period of 3 weeks. Strikingly, wild type mice treated with eitherXBP1- or IRE-1-silencing nanoparticles demonstrated a remarkableincrease in survival compared with control groups (FIG. 14I). Mostimportantly, Rag2-deficient hosts bearing ovarian tumors were totallyunable to respond to this treatment, demonstrating that an intactadaptive immune system is necessary for the observed therapeutic benefit(FIG. 14J). Together, these data demonstrate that targeting theIRE-1α/XBP1 branch of the ER stress response in tDCs induces protectiveanti-tumor immune responses against otherwise lethal ovarian cancer.

TABLE 1 Primers Species Gene Oligo name Sequence 5′-3′ Purpose HUMANACTB hAct-F GCGAGAAGATGACCCAGATC RT-qPCR hAct-R CCAGTGGTACGGCCAGAGGHUMAN DDIT3/ hCHOP-F CTGCTTCTCTGGCTTGGCTG RT-qPCR CHOP hCHOP-RGCTCTGGGAGGTGCTTGTGA HUMAN XBP1 hXBP1-SA-F CCTGGTTGCTGAAGAGGAGG SplicinghXBP1-SA-R CCATGGGGAGTTCTGGAG Assay Mouse Xbp1 Xbp1-SA-FACACGTTTGGGAATGGACAC Splicing Xbp1-SA-F CCATGGGAAGATGTTCTGGG Assay MouseActb actb1083 CTCAGGAGGAGCAATGATCTTGAT RT-qPCR actb987TACCACCATGTACCCAGGCA Mouse Xbp1 Xbp1.total-F GACAGAGAGTCAAACTAACGTGGRT-qPCR Xbp1.total-R GTCCAGCAGGCAAGAAGGT Mouse Xbp1s XBPsA406FAAGAACACGCTTGGGAATGG RT-qPCR XBPsAa518R CTGCACCTGCTGCGGAC Mouse Xbp1XBP1WT205-F CCTGAGCCCGGAGGAGAA RT-qPCR (exon 2) XBP1WT272-RCTCGAGCAGTCTGCGCTG (does not amplify deleted exon 2) Mouse Dnajb9/ERdj4-F TAAAAGCCCTGATGCTGAAGC RT-qPCR Erdj4 ERdj4-R TCCGACTATTGGCATCCGAMouse Sec61a1 Sec61a1-F CTATTTCCAGGGCTTCCGAGT RT-qPCR Sec61a1-RAGGTGTTGTACTGGCCTCGGT Mouse Hspa5/ Grp78-F TCATCGGACGCACTTGGAA RT-qPCRPiP Grp78-R CAACCACCTTGAATGGCAAGA Mouse Ddit3/ CHOP-FGTCCCTAGCTTGGCTGACAGA RT-qPCR CHOP CHOP-R TGGAGAGCGAGGGCTTTG MouseAgpat6 Agpat6-F AGCTTGATTGTCAACCTCCTG RT-qPCR Agpat6-RCCGTTGGTGTAGGGCTTGT Mouse Scd2 Scd2-F GCATTTGGGAGCCTTGTACG RT-qPCRScd2-R AGCCGTGCCTTGTATGTTCTG Mouse Fasn Fasn-F GGAGGTGGTGATAGCCGGTATRT-qPCR Fasn-R TGGGTAATCCATAGAGCCCAG Mouse Lpar1 Lpar1.FGACCTAGCAGGCTTACAGTTCC RT-qPCR Lpar1.R GCTGTAGTTTGGGGCGATGA Mouse Clec9aClec9a.F GAGCATGGTGTGTTGTGACG RT-qPCR Clec9a.R TACCTGGAAGAACTTGATGCCCMouse Zbtb46 Zbtb46.F CTCACATACTGGAGAGCGGC RT-qPCR Zbtb46.RTGCTGTGGACCAGAGTATGTC

TABLE 2 KO/WT Symbol Description fold Fut7 fucosyltransferase 7 −2.32Slc5a3 solute carrier family 5 (inositol −2.22 transporters), member 3Gm2a GM2 ganglioside activator protein −1.99 Stt3a STT3, subunit of the−1.94 oligosaccharyltransferase complex, homolog A (S. cerevisiae) Uggt1UDP-glucose glycoprotein −1.81 glucosyltransferase 1 Gfpt1 glutaminefructose-6-phosphate −1.77 transaminase 1 Mlec malectin −1.73 Glceglucuronyl C5-epimerase −1.68 Hk1 hexokinase 1 −1.59 Aldoc aldolase C,fructose-bisphosphate −1.58 Rpn1 ribophorin I −1.58 Mgat1 mannosideacetylglucosaminyltransferase 1 −1.56 Pfkp phosphofructokinase, platelet−1.56 Gaa glucosidase, alpha, acid −1.55 Phkb phosphorylase kinase beta−1.55 Ogdh oxoglutarate (alpha-ketoglutarate) −1.52 dehydrogenase(lipoamide) Ptafr platelet-activating factor receptor −1.52 Pgm3phosphoglucomutase 3 −1.52 Pygb brain glycogen phosphorylase −1.51 Iduaiduronidase, alpha-L- −1.49 Acly ATP citrate lyase −1.48 Ppip5k2diphosphoinositol pentakisphosphate −1.47 kinase 2 Cpt1a carnitinepalmitoyltransferase 1a, liver −1.47 Pkm pyruvate kinase, muscle −1.46St3gal4 ST3 beta-galactoside alpha-2,3- −1.45 sialyltransferase 4 Ganabalpha glucosidase 2 alpha neutral subunit −1.45 Man2b1 mannosidase 2,alpha B1 −1.43 Rpn2 ribophorin II −1.40 Tram2 translocatingchain-associating −6.39 membrane protein 2 Zfp781 zinc finger protein781 −4.18 Apol9b apolipoprotein L 9b −4.08 Tlr12 toll-like receptor 12−4.06 Zfp791 zinc finger protein 791 −3.93 9530036M11Rik RIKEN cDNA9530036M11 gene −3.71 Leprel1 leprecan-like 1 −3.29 Il12b interleukin12b −3.12 Scn4b sodium channel, type IV, beta −3.08 Tuba1c tubulin,alpha 1C −3.00 Dkk3 dickkopf homolog 3 (Xenopus laevis) −2.98 Sardhsarcosine dehydrogenase −2.96 Cd226 CD226 antigen −2.90 Synpo2synaptopodin 2 −2.87 Fbxo10 F-box protein 10 −2.79 Rtkn rhotekin −2.75Fam13c family with sequence similarity 13, −2.72 member C Itgb2 integrinbeta 2 −2.62 Ptgir prostaglandin I receptor (IP) −2.61 Bcorl1 BCL6co-repressor-like 1 −2.54 Rnase2a #N/A −2.53 Fcrls Fc receptor-like S,scavenger receptor −2.47 Hyou1 hypoxia up-regulated 1 −2.47 Pdia5protein disulfide isomerase associated 5 −2.45 Sdc1 syndecan 1 −2.42Tmem25 transmembrane protein 25 −2.40 Ctnna2 catenin (cadherinassociated protein), −2.39 alpha 2 F7 coagulation factor VII −2.37 Itgaeintegrin alpha E, epithelial-associated −2.35 Extl1 exostoses(multiple)-like 1 −2.28 Spryd3 SPRY domain containing 3 −2.26 Rps6kc1ribosomal protein S6 kinase polypeptide 1 −2.26 Ear2eosinophil-associated, ribonuclease A −2.24 family, member 2 Fn1fibronectin 1 −2.23 Tenm4 teneurin transmembrane protein 4 −2.22 Gfra2glial cell line derived neurotrophic factor −2.21 family receptor alpha2 Plekhn1 pleckstrin homology domain containing, −2.21 family N member 1Lrrc18 leucine rich repeat containing 18 −2.20 Nucb2 nucleobindin 2−2.20 Cacna1d calcium channel, voltage-dependent, L −2.16 type, alpha 1Dsubunit Kctd12b potassium channel tetramerisation −2.16 domaincontaining 12b Hspa14 heat shock protein 14 −2.16 Galnt9UDP-N-acetyl-alpha-D- −2.13 galactosamine:polypeptide N-acetylgalactosaminyltransferase 9 Hdlbp high density lipoprotein (HDL)binding −2.11 protein Snx22 sorting nexin 22 −2.10 Dnmbp dynamin bindingprotein −2.05 Gm8221 apolipoprotein L 7c pseudogene −2.04 Lpar1lysophosphatidic acid receptor 1 −2.04 Sec61a1 Sec61 alpha 1 subunit (S.cerevisiae) −2.03 Nlrx1 NLR family member X1 −2.03 Capn5 calpain 5 −2.03Retnla resistin like alpha −2.02 Sec24d Sec24 related gene family,member D (S. cerevisiae) −2.02 Atg9b autophagy related 9B −2.00 Mpzl2myelin protein zero-like 2 −1.97 Slc13a3 solute carrier family 13(sodium- −1.97 dependent dicarboxylate transporter), member 3 Plce1phospholipase C, epsilon 1 −1.97 Otof otoferlin −1.96 F10 coagulationfactor X −1.96 Unc45b unc-45 homolog B (C. elegans) −1.96 Hspa13 heatshock protein 70 family, member 13 −1.93 Madd MAP-kinase activatingdeath domain −1.92 Nbas neuroblastoma amplified sequence −1.92 Sec24cSec24 related gene family, member C (S. cerevisiae) −1.91 Ercc6lexcision repair cross-complementing −1.90 rodent repair deficiencycomplementation group 6 like 1700021K19Rik RIKEN cDNA 1700021K19 gene−1.88 Abca3 ATP-binding cassette, sub-family A −1.88 (ABC1), member 3Kctd11 potassium channel tetramerisation −1.88 domain containing 11Agpat4 1-acylglycerol-3-phosphate O- −1.87 acyltransferase 4(lysophosphatidic acid acyltransferase, delta) Ambra1 autophagy/beclin 1regulator 1 −1.87 Xdh xanthine dehydrogenase −1.86 Surf4 surfeit gene 4−1.86 Pltp phospholipid transfer protein −1.86 Itgam integrin alpha M−1.86 Cldn1 claudin 1 −1.85 Atg13 autophagy related 13 −1.84 Xbp1 X-boxbinding protein 1 −1.83 P4hb prolyl 4-hydroxylase, beta polypeptide−1.83 Cdk5rap3 CDK5 regulatory subunit associated −1.83 protein 3 GmppbGDP-mannose pyrophosphorylase B −1.83 Zfp872 zinc finger protein 872−1.82 Zfp949 zinc finger protein 949 −1.82 Psd4 pleckstrin and Sec7domain containing 4 −1.81 Pidd1 #N/A −1.80 Dock2 dedicator ofcyto-kinesis 2 −1.80 Sidt2 SID1 transmembrane family, member 2 −1.804931406H21Rik RIKEN cDNA 4931406H21 gene −1.79 Abca9 ATP-bindingcassette, sub-family A −1.79 (ABC1), member 9 Ppm1f protein phosphatase1F (PP2C domain −1.79 containing) Abcg3 ATP-binding cassette, sub-familyG −1.79 (WHITE), member 3 Crat carnitine acetyltransferase −1.78 Gpr56 Gprotein-coupled receptor 56 −1.78 Bub1b budding uninhibited bybenzimidazoles 1 −1.77 homolog, beta (S. cerevisiae) Ticam1 toll-likereceptor adaptor molecule 1 −1.77 Il27ra interleukin 27 receptor, alpha−1.77 Fam129a family with sequence similarity 129, −1.77 member ADnase1l3 deoxyribonuclease 1-like 3 −1.76 Ptger2 prostaglandin Ereceptor 2 (subtype EP2) −1.75 Gm1966 predicted gene 1966 −1.75 Tmem167btransmembrane protein 167B −1.75 Nup188 nucleoporin 188 −1.75 Tyk2tyrosine kinase 2 −1.75 Asb4 ankyrin repeat and SOCS box-containing 4−1.74 Tom1l2 target of myb1-like 2 (chicken) −1.74 Ascc2 activatingsignal cointegrator 1 complex −1.74 subunit 2 Clec4a2 C-type lectindomain family 4, member a2 −1.73 Polr3b polymerase (RNA) III (DNAdirected) −1.73 polypeptide B Igj immunoglobulin joining chain −1.73Acot11 acyl-CoA thioesterase 11 −1.73 Ankrd23 ankyrin repeat domain 23−1.73 Mis12 MIS12 homolog (yeast) −1.73 Rfwd3 ring finger and WD repeatdomain 3 −1.73 Ccpg1 cell cycle progression 1 −1.72 F2rl2 coagulationfactor II (thrombin) receptor- −1.71 like 2 Arfgap3 ADP-ribosylationfactor GTPase −1.71 activating protein 3 Mmp25 matrix metallopeptidase25 −1.71 Ap1g1 adaptor protein complex AP-1, gamma 1 −1.70 subunit Cenpicentromere protein I −1.70 Tlr8 toll-like receptor 8 −1.70 Pdcd1programmed cell death 1 −1.69 Tmem127 transmembrane protein 127 −1.69Plau plasminogen activator, urokinase −1.69 Tut1 terminal uridylyltransferase 1, U6 −1.69 snRNA-specific Mical1 microtubule associatedmonooxygenase, −1.69 calponin and LIM domain containing 1 Mrc1 mannosereceptor, C type 1 −1.69 Pdia4 protein disulfide isomerase associated 4−1.69 Zfp738 zinc finger protein 738 −1.69 Kat6a K(lysine)acetyltransferase 6A −1.69 Mela melanoma antigen −1.69 Mettl14methyltransferase like 14 −1.68 Copg1 coatomer protein complex, subunit−1.68 gamma 1 Rassf4 Ras association (RaIGDS/AF-6) domain −1.68 familymember 4 Klhl18 kelch-like 18 −1.68 Gorab golgin, RAB6-interacting −1.68Rab8b RAB8B, member RAS oncogene family −1.67 Spcs3 signal peptidasecomplex subunit 3 −1.67 homolog (S. cerevisiae) Btd biotinidase −1.67Pias3 protein inhibitor of activated STAT 3 −1.67 Lysmd3 LysM, putativepeptidoglycan-binding, −1.67 domain containing 3 Vps8 vacuolar proteinsorting 8 homolog (S. cerevisiae) −1.67 Calu calumenin −1.67 Sdccag8serologically defined colon cancer antigen 8 −1.66 Gpr68 Gprotein-coupled receptor 68 −1.66 Kcnab2 potassium voltage-gatedchannel, shaker- −1.66 related subfamily, beta member 2 Tlr11 toll-likereceptor 11 −1.66 Slc39a7 solute carrier family 39 (zinc transporter),−1.66 member 7 Fasn fatty acid synthase −1.65 E2f1 E2F transcriptionfactor 1 −1.65 Zc3h12c zinc finger CCCH type containing 12C −1.65 Fosl2fos-like antigen 2 −1.65 Mcph1 microcephaly, primary autosomal −1.64recessive 1 Tgoln2 trans-golgi network protein 2 −1.64 Dpp7dipeptidylpeptidase 7 −1.64 Golga3 golgi autoantigen, golgin subfamilya, 3 −1.63 Far1 fatty acyl CoA reductase 1 −1.63 Pank4 pantothenatekinase 4 −1.63 Ncapd3 non-SMC condensin II complex, subunit −1.63 D3Ddb1 damage specific DNA binding protein 1 −1.62 Map3k14mitogen-activated protein kinase kinase −1.62 kinase 14 Surf6 surfeitgene 6 −1.62 Sulf1 sulfatase 1 −1.62 Samd9l sterile alpha motif domaincontaining 9- −1.62 like Slc41a2 solute carrier family 41, member 2−1.62 Tnip2 TNFAIP3 interacting protein 2 −1.62 Klhl28 kelch-like 28−1.62 Lclat1 lysocardiolipin acyltransferase 1 −1.62 Peg13 paternallyexpressed 13 −1.61 Crem cAMP responsive element modulator −1.61 Mmp19matrix metallopeptidase 19 −1.61 Copa coatomer protein complex subunitalpha −1.61 Rin3 Ras and Rab interactor 3 −1.61 Ldlr low densitylipoprotein receptor −1.61 Nxpe4 neurexophilin and PC-esterase domain−1.61 family, member 4 Dock10 dedicator of cytokinesis 10 −1.61 Leprotleptin receptor overlapping transcript −1.61 Sept9 septin 9 −1.61 Fbxo38F-box protein 38 −1.61 Myo1f myosin IF −1.60 Znfx1 zinc finger,NFX1-type containing 1 −1.60 Ddi2 DNA-damage inducible protein 2 −1.60Anpep alanyl (membrane) aminopeptidase −1.60 Lifr leukemia inhibitoryfactor receptor −1.60 Utp20 UTP20, small subunit (SSU) processome −1.60component, homolog (yeast) Lpin1 lipin 1 −1.60 Eps8 epidermal growthfactor receptor pathway −1.60 substrate 8 Zfyve16 zinc finger, FYVEdomain containing 16 −1.60 Arhgap18 Rho GTPase activating protein 18−1.60 Elmo2 engulfment and cell motility 2 −1.60 Daglb diacylglycerollipase, beta −1.59 Elmod2 ELMO/CED-12 domain containing 2 −1.59 Abcd1ATP-binding cassette, sub-family D −1.59 (ALD), member 1 Alpk1alpha-kinase 1 −1.59 Ncs1 neuronal calcium sensor 1 −1.59 Ticam2toll-like receptor adaptor molecule 2 −1.59 Clasp1 CLIP associatingprotein 1 −1.59 Atxn3 ataxin 3 −1.59 Depdc5 DEP domain containing 5−1.58 Cnnm4 cyclin M4 −1.58 Steap3 STEAP family member 3 −1.58 Kit kitoncogene −1.58 1110037F02Rik RIKEN cDNA 1110037F02 gene −1.58 Supt5suppressor of Ty 5 −1.58 Socs6 suppressor of cytokine signaling 6 −1.58Gm5431 predicted gene 5431 −1.57 Nup160 nucleoporin 160 −1.57 Atf6activating transcription factor 6 −1.57 Net1 neuroepithelial celltransforming gene 1 −1.57 Dhdh dihydrodiol dehydrogenase (dimeric) −1.57Dapk1 death associated protein kinase 1 −1.57 Taf1 TAF1 RNA polymeraseII, TATA box −1.57 binding protein (TBP)-associated factor Arhgap11a RhoGTPase activating protein 11A −1.57 Supt16 suppressor of Ty 16 −1.57Dnajc3 DnaJ (Hsp40) homolog, subfamily C, −1.57 member 3 Ddx10 DEAD(Asp-Glu-Ala-Asp) box polypeptide −1.56 10 Gm8995 predicted gene 8995−1.56 Bsdc1 BSD domain containing 1 −1.56 Gpr108 G protein-coupledreceptor 108 −1.56 Inpp5d inositol polyphosphate-5-phosphatase D −1.56Snrnp200 small nuclear ribonucleoprotein 200 (U5) −1.56 Asb6 ankyrinrepeat and SOCS box-containing 6 −1.56 Mfn2 mitofusin 2 −1.56 Ccr2chemokine (C-C motif) receptor 2 −1.56 Cbl Casitas B-lineage lymphoma−1.56 Mrvi1 MRV integration site 1 −1.56 Tap2 transporter 2, ATP-bindingcassette, sub- −1.56 family B (MDR/TAP) Mcm3 minichromosome maintenancedeficient 3 −1.56 (S. cerevisiae) Nol8 nucleolar protein 8 −1.56 Gltpd1glycolipid transfer protein domain −1.56 containing 1 Plekho2 pleckstrinhomology domain containing, −1.56 family O member 2 Il1rl1 interleukin 1receptor-like 1 −1.56 Ankfy1 ankyrin repeat and FYVE domain −1.56containing 1 Gpatch8 G patch domain containing 8 −1.55 Nap1l4 nucleosomeassembly protein 1-like 4 −1.55 Tmed9 transmembrane emp24 proteintransport −1.55 domain containing 9 Arhgap26 Rho GTPase activatingprotein 26 −1.55 Uspl1 ubiquitin specific peptidase like 1 −1.55 Pdia6protein disulfide isomerase associated 6 −1.55 Arrb1 arrestin, beta 1−1.55 Mkl1 MKL (megakaryoblastic −1.55 leukemia)/myocardin-like 1 Zfmlzinc finger, matrin-like −1.55 Fchsd2 FCH and double SH3 domains 2 −1.54Cnr2 cannabinoid receptor 2 (macrophage) −1.54 Ccdc47 coiled-coil domaincontaining 47 −1.54 Dok2 docking protein 2 −1.54 Taok3 TAO kinase 3−1.54 Tti1 TELO2 interacting protein 1 −1.54 Smc5 structural maintenanceof chromosomes 5 −1.54 Pbxip1 pre B cell leukemia transcription factor−1.54 interacting protein 1 Prr5l proline rich 5 like −1.54 Stat6 signaltransducer and activator of −1.54 transcription 6 Hepacam2 HEPACAMfamily member 2 −1.54 2700049A03Rik RIKEN cDNA 2700049A03 gene −1.54Pkd1 polycystic kidney disease 1 homolog −1.54 Lrrk1 leucine-rich repeatkinase 1 −1.54 Pan2 PAN2 polyA specific ribonuclease subunit −1.54homolog (S. cerevisiae) Magt1 magnesium transporter 1 −1.53 Armcx3armadillo repeat containing, X-linked 3 −1.53 Cd1d1 CD1d1 antigen −1.53Plekhb2 pleckstrin homology domain containing, −1.53 family B (evectins)member 2 Bcl2l14 BCL2-like 14 (apoptosis facilitator) −1.53 Lmf2 lipasematuration factor 2 −1.53 Nhsl2 NHS-like 2 −1.53 Timp3 tissue inhibitorof metalloproteinase 3 −1.53 Casp3 caspase 3 −1.53 NaaaN-acylethanolamine acid amidase −1.52 Nfam1 Nfat activating moleculewith ITAM motif 1 −1.52 Dcp1a DCP1 decapping enzyme homolog A (S.cerevisiae) −1.52 Eif3a eukaryotic translation initiation factor 3,−1.52 subunit A Mtmr6 myotubularin related protein 6 −1.52 Top2atopoisomerase (DNA) II alpha −1.52 Dnajc14 DnaJ (Hsp40) homolog,subfamily C, −1.52 member 14 Pde4dip phosphodiesterase 4D interactingprotein −1.52 (myomegalin) Evi2a ecotropic viral integration site 2a−1.52 Npat nuclear protein in the AT region −1.51 Sil1 endoplasmicreticulum chaperone SIL1 −1.51 homolog (S. cerevisiae) Hltfhelicase-like transcription factor −1.51 Stim2 stromal interactionmolecule 2 −1.51 Baz2a bromodomain adjacent to zinc finger −1.51 domain,2A EII elongation factor RNA polymerase II −1.51 R3hcc1l R3H domain andcoiled-coil containing 1 −1.51 like Mybbp1a MYB binding protein (P160)1a −1.51 Myo1c myosin IC −1.50 Zfp260 zinc finger protein 260 −1.50Adam19 a disintegrin and metallopeptidase −1.50 domain 19 (meltrin beta)Tkt transketolase −1.50 Exoc2 exocyst complex component 2 −1.50 Sft2d2SFT2 domain containing 2 −1.50 Tuba1a tubulin, alpha 1A −1.50 Oatornithine aminotransferase −1.50 Baz2b bromodomain adjacent to zincfinger −1.50 domain, 2B Ltn1 listerin E3 ubiquitin protein ligase 1−1.50 Copb2 coatomer protein complex, subunit beta 2 −1.50 (beta prime)Cyp51 cytochrome P450, family 51 −1.50 Acin1 apoptotic chromatincondensation inducer 1 −1.50 Hivep2 human immunodeficiency virus type I−1.50 enhancer binding protein 2 Tmem170b transmembrane protein 170B−1.49 Stom stomatin −1.49 Sec16a SEC16 homolog A (S. cerevisiae) −1.49Krt80 keratin 80 −1.49 Malat1 metastasis associated lung −1.49adenocarcinoma transcript 1 (non-coding RNA) Rabgap1 RAB GTPaseactivating protein 1 −1.49 Pja2 praja 2, RING-H2 motif containing −1.49Slamf8 SLAM family member 8 −1.49 Tnpo3 transportin 3 −1.48 Tmx3thioredoxin-related transmembrane −1.48 protein 3 Rbbp6 retinoblastomabinding protein 6 −1.48 Ilf3 interleukin enhancer binding factor 3 −1.48Ctr9 Ctr9, Paf1/RNA polymerase II complex −1.48 component, homolog (S.cerevisiae) Anapc1 anaphase promoting complex subunit 1 −1.48 Svilsupervillin −1.48 Ptrf polymerase I and transcript release factor −1.48Tox4 TOX high mobility group box family −1.48 member 4 Abi2abl-interactor 2 −1.48 Atp7a ATPase, Cu++ transporting, alpha −1.48polypeptide Cnppd1 cyclin Pas1/PHO80 domain containing 1 −1.48 Zc3h7azinc finger CCCH type containing 7 A −1.48 Zfp263 zinc finger protein263 −1.48 Ipo5 importin 5 −1.48 Pggt1b protein geranylgeranyltransferasetype I, −1.48 beta subunit Ptplad2 protein tyrosine phosphatase-like A−1.48 domain containing 2 Usp8 ubiquitin specific peptidase 8 −1.48Coro7 coronin 7 −1.47 Sept3 septin 3 −1.47 Spop speckle-type POZ protein−1.47 Aco1 aconitase 1 −1.47 Nucb1 nucleobindin 1 −1.47 Prpf6 PRP6pre-mRNA splicing factor 6 −1.47 homolog (yeast) Mavs mitochondrialantiviral signaling protein −1.47 Cd209c CD209c antigen −1.47 Atp8b4ATPase, class I, type 8B, member 4 −1.47 Myof myoferlin −1.47 Sec23bSEC23B (S. cerevisiae) −1.46 Nckap1l NCK associated protein 1 like −1.46Pum1 pumilio RNA-binding family member 1 −1.46 Fgl2 fibrinogen-likeprotein 2 −1.46 Sec31a Sec31 homolog A (S. cerevisiae) −1.46 Txndc16thioredoxin domain containing 16 −1.46 Pdxk pyridoxal (pyridoxine,vitamin B6) kinase −1.46 Gga3 golgi associated, gamma adaptin ear −1.46containing, ARF binding protein 3 Kitl kit ligand −1.46 Tmcc3transmembrane and coiled coil domains 3 −1.46 Gmppa GDP-mannosepyrophosphorylase A −1.46 Ppp1r21 protein phosphatase 1, regulatorysubunit −1.46 21 Os9 amplified in osteosarcoma −1.46 4930506M07Rik RIKENcDNA 4930506M07 gene −1.45 Smc1a structural maintenance of chromosomes−1.45 1A Kansl3 KAT8 regulatory NSL complex subunit 3 −1.452900097C17Rik RIKEN cDNA 2900097C17 gene −1.45 Tmem181b-ps transmembraneprotein 181B, −1.45 pseudogene Nup210 nucleoporin 210 −1.45 Entpd7ectonucleoside triphosphate −1.45 diphosphohydrolase 7 Stag2 stromalantigen 2 −1.45 Narf nuclear prelamin A recognition factor −1.45 Tmem39atransmembrane protein 39a −1.45 Rnf41 ring finger protein 41 −1.45Agpat6 1-acylglycerol-3-phosphate O- −1.44 acyltransferase 6(lysophosphatidic acid acyltransferase, zeta) Zfp445 zinc finger protein445 −1.44 Ikbkg inhibitor of kappaB kinase gamma −1.44 Gripap1 GRIP1associated protein 1 −1.44 Chd2 chromodomain helicase DNA binding −1.44protein 2 Ankrd27 ankyrin repeat domain 27 (VPS9 domain) −1.44 Ppp1r15bprotein phosphatase 1, regulatory −1.44 (inhibitor) subunit 15b Golgb1golgi autoantigen, golgin subfamily b, −1.44 macrogolgin 1 Tmed3transmembrane emp24 domain −1.44 containing 3 Psap prosaposin −1.44Ctnna1 catenin (cadherin associated protein), −1.44 alpha 1 Ptbp1polypyrimidine tract binding protein 1 −1.43 Csf2rb colony stimulatingfactor 2 receptor, beta, −1.43 low-affinity (granulocyte-macrophage)Ssrp1 structure specific recognition protein 1 −1.43 Cyth4 cytohesin 4−1.43 Tug1 taurine upregulated gene 1 −1.43 Scd2 stearoyl-Coenzyme Adesaturase 2 −1.43 Arhgef6 Rac/Cdc42 guanine nucleotide exchange −1.43factor (GEF) 6 Tbc1d15 TBC1 domain family, member 15 −1.42 Mbnl1muscleblind-like 1 (Drosophila) −1.42 Eif4g2 eukaryotic translationinitiation factor 4, −1.42 gamma 2 Rp2h retinitis pigmentosa 2 homolog(human) −1.42 Adam8 a disintegrin and metallopeptidase −1.41 domain 8Neat1 nuclear paraspeckle assembly transcript −1.41 1 (non-proteincoding) Themis2 thymocyte selection associated family −1.41 member 2Gosr2 golgi SNAP receptor complex member 2 −1.40 Idi1isopentenyl-diphosphate delta isomerase −1.40 Psmd2 proteasome (prosome,macropain) 26S −1.40 subunit, non-ATPase, 2 Hmgcr3-hydroxy-3-methylglutaryl-Coenzyme A −1.40 reductase Eef2 eukaryotictranslation elongation factor 2 −1.40 Myo1g myosin IG −1.40 Msn moesin−1.39 Cfh complement component factor h −1.38 C1qc complement component1, q 1.42 subcomponent, C chain A630089N07Rik RIKEN cDNA A630089N07 gene1.43 Traf5 TNF receptor-associated factor 5 1.44 Zfp353-ps zinc fingerprotein 352 1.45 Fam110b family with sequence similarity 110, 1.48member B Wfdc17 WAP four-disulfide core domain 17 1.48 Fam213b familywith sequence similarity 213, 1.48 member B Trem2 triggering receptorexpressed on myeloid 1.50 cells 2 Snx18 sorting nexin 18 1.511500012F01Rik RIKEN cDNA 1500012F01 gene 1.51 Tctex1d2 Tctex1 domaincontaining 2 1.52 Icam2 intercellular adhesion molecule 2 1.53 Gngt2guanine nucleotide binding protein (G 1.53 protein), gamma transducingactivity polypeptide 2 Lst1 leukocyte specific transcript 1 1.54 Itsn1intersectin 1 (SH3 domain protein 1A) 1.54 Ltbp1 latent transforminggrowth factor beta 1.55 binding protein 1 Blvrb biliverdin reductase B(flavin reductase 1.57 (NADPH)) Hscb HscB iron-sulfur clusterco-chaperone 1.57 homolog (E. coli) Acot2 acyl-CoA thioesterase 2 1.57Gimap8 GTPase, IMAP family member 8 1.59 Sept1 septin 1 1.60 C5ar2complement component 5a receptor 2 1.60 Itga6 integrin alpha 6 1.61 Dcxrdicarbonyl L-xylulose reductase 1.61 Mmp12 matrix metallopeptidase 121.61 Tmem243 transmembrane protein 243, 1.63 mitochondrial Pou2f2 POUdomain, class 2, transcription factor 2 1.63 Ly6c2 lymphocyte antigen 6complex, locus C2 1.63 Hnmt histamine N-methyltransferase 1.64 Asnsasparagine synthetase 1.64 Emp1 epithelial membrane protein 1 1.65 Ets1E26 avian leukemia oncogene 1, 5′ 1.65 domain Cirbp cold inducible RNAbinding protein 1.65 Cbr2 carbonyl reductase 2 1.68 Serpinb10 serine (orcysteine) peptidase inhibitor, 1.68 clade B (ovalbumin), member 10 Vsig4V-set and immunoglobulin domain 1.69 containing 4 Fam20c family withsequence similarity 20, 1.69 member C Irf2bpl interferon regulatoryfactor 2 binding 1.71 protein-like Cd93 CD93 antigen 1.71 Med17 mediatorcomplex subunit 17 1.71 Apoe apolipoprotein E 1.72 Cd28 CD28 antigen1.72 Ms4a1 membrane-spanning 4-domains, 1.73 subfamily A, member 1Lpcat2 lysophosphatidylcholine acyltransferase 2 1.73 Plac8placenta-specific 8 1.73 Rab28 RAB28, member RAS oncogene family 1.74Mzb1 marginal zone B and B1 cell-specific 1.74 protein 1 Dok3 dockingprotein 3 1.75 Arg1 arginase, liver 1.76 Cd19 CD19 antigen 1.76 Fcnaficolin A 1.77 Cd79b CD79B antigen 1.77 Tspan4 tetraspanin 4 1.78 Mmp9matrix metallopeptidase 9 1.80 Itk IL2 inducible T cell kinase 1.81Dppa3 developmental pluripotency-associated 3 1.81 Ninj1 ninjurin 1 1.81Cxcr5 chemokine (C—X—C motif) receptor 5 1.82 Spry2 sprouty homolog 2(Drosophila) 1.82 Prtn3 proteinase 3 1.82 Snx24 sorting nexing 24 1.83Ccl3 chemokine (C-C motif) ligand 3 1.83 Gdf3 growth differentiationfactor 3 1.86 Ly6d lymphocyte antigen 6 complex, locus D 1.86 Cd69 CD69antigen 1.86 Selp selectin, platelet 1.86 Apoc2 apolipoprotein C-II 1.86Gimap3 GTPase, IMAP family member 3 1.88 Speer7-ps1 spermatogenesisassociated glutamate 1.89 (E)-rich protein 7, pseudogene 1 Dusp1 dualspecificity phosphatase 1 1.89 Clec4d C-type lectin domain family 4,member d 1.90 Cd27 CD27 antigen 1.90 Sh2d2a SH2 domain protein 2A 1.91Ly6a lymphocyte antigen 6 complex, locus A 1.92 Nt5e 5′ nucleotidase,ecto 1.94 Txk TXK tyrosine kinase 1.96 Saa3 serum amyloid A 3 1.96Gimap9 GTPase, IMAP family member 9 1.98 Ndufb2 NADH dehydrogenase(ubiquinone) 1 1.99 beta subcomplex, 2 Tfec transcription factor EC 2.02Cxcl13 chemokine (C—X—C motif) ligand 13 2.03 Igfbp4 insulin-like growthfactor binding protein 4 2.04 Asb1 ankyrin repeat and SOCSbox-containing 1 2.07 Pf4 platelet factor 4 2.07 Xkrx X Kell blood groupprecursor related X 2.09 linked Ypel3 yippee-like 3 (Drosophila) 2.09Ppp3cc protein phosphatase 3, catalytic subunit, 2.10 gamma isoformFam101b family with sequence similarity 101, 2.10 member B Slc40a1solute carrier family 40 (iron-regulated 2.10 transporter), member 1Samd3 sterile alpha motif domain containing 3 2.11 Il2rb interleukin 2receptor, beta chain 2.12 Fam169b family with sequence similarity 169,2.12 member B Mri1 methylthioribose-1-phosphate isomerase 2.15 homolog(S. cerevisiae) Gimap6 GTPase, IMAP family member 6 2.15 Alox15arachidonate 15-lipoxygenase 2.17 Tpsb2 tryptase beta 2 2.18 Lef1lymphoid enhancer binding factor 1 2.18 Tcf7 transcription factor 7, Tcell specific 2.21 Skap1 src family associated phosphoprotein 1 2.22Tmprss13 transmembrane protease, serine 13 2.22 Marco macrophagereceptor with collagenous 2.23 structure Cd79a CD79A antigen(immunoglobulin- 2.26 associated alpha) Apoc1 apolipoprotein C-I 2.26Ikzf3 IKAROS family zinc finger 3 2.27 Cd3d CD3 antigen, deltapolypeptide 2.29 Id1 inhibitor of DNA binding 1 2.29 Gzmb granzyme B2.29 Satb1 special AT-rich sequence binding protein 1 2.29 Lax1lymphocyte transmembrane adaptor 1 2.32 Gimap4 GTPase, IMAP familymember 4 2.34 Cpa3 carboxypeptidase A3, mast cell 2.36 Ccr3 chemokine(C-C motif) receptor 3 2.37 Fam189b family with sequence similarity 189,2.38 member B Wnt2 wingless-related MMTV integration site 2 2.38 Vmn2r26vomeronasal 2, receptor 26 2.39 Ppbp pro-platelet basic protein 2.40Pou2af1 POU domain, class 2, associating factor 1 2.41 Lck lymphocyteprotein tyrosine kinase 2.42 Gm11346 X-linked lymphocyte-regulated 52.42 pseudogene Cma1 chymase 1, mast cell 2.42 Ctsw cathepsin W 2.43Gm684 predicted gene 684 2.45 Serpinb2 serine (or cysteine) peptidaseinhibitor, 2.47 clade B, member 2 Trav3n-3 T cell receptor alphavariable 3N-3 2.48 Prg4 proteoglycan 4 (megakaryocyte 2.48 stimulatingfactor, articular superficial zone protein) Slpi secretory leukocytepeptidase inhibitor 2.49 Tmem51os1 Tmem51 opposite strand 1 2.50 Ccl4chemokine (C-C motif) ligand 4 2.50 Klre1 killer cell lectin-likereceptor family E 2.51 member 1 Pear1 platelet endothelial aggregationreceptor 1 2.52 Lat linker for activation of T cells 2.53 Ptchd4 patcheddomain containing 4 2.53 Pard6g par-6 family cell polarity regulatorgamma 2.55 Prf1 perforin 1 (pore forming protein) 2.57 Stc1stanniocalcin 1 2.59 Scn1a sodium channel, voltage-gated, type I, 2.60alpha Plcb4 phospholipase C, beta 4 2.63 Cd3g CD3 antigen, gammapolypeptide 2.67 Gpr174 G protein-coupled receptor 174 2.69 Pik3c2bphosphoinositide-3-kinase, class 2, beta 2.71 polypeptide Crtamcytotoxic and regulatory T cell molecule 2.71 Eomes eomesodermin homolog(Xenopus laevis) 2.72 Ebf1 early B cell factor 1 2.72 Tnfsf15 tumornecrosis factor (ligand) 2.77 superfamily, member 15 Gimap7 GTPase, IMAPfamily member 7 2.81 Ifitm10 interferon induced transmembrane 2.82protein 10 Trem3 triggering receptor expressed on myeloid 2.83 cells 3Fasl Fas ligand (TNF superfamily, member 6) 2.83 Epha2 Eph receptor A22.83 Sh2d1a SH2 domain protein 1A 2.85 Tdgf1 teratocarcinoma-derivedgrowth factor 1 2.86 Ncr1 natural cytotoxicity triggering receptor 12.88 Fbxl21 F-box and leucine-rich repeat protein 21 2.88 Zdhhc15 zincfinger, DHHC domain containing 15 2.89 Gimap5 GTPase, IMAP family member5 2.91 Gzma granzyme A 2.94 A630023P12Rik RIKEN cDNA A630023P12 gene2.96 Ms4a4b membrane-spanning 4-domains, 2.99 subfamily A, member 4BMgst2 microsomal glutathione S-transferase 2 2.99 Thy1 thymus cellantigen 1, theta 2.99 2900011O08Rik RIKEN cDNA 2900011O08 gene 3.01 Cd3eCD3 antigen, epsilon polypeptide 3.02 A730060N03Rik RIKEN cDNAA730060N03 gene 3.03 Klrc2 killer cell lectin-like receptor subfamily C,3.04 member 2 Phactr3 phosphatase and actin regulator 3 3.10 Retnlgresistin like gamma 3.11 Tsix X (inactive)-specific transcript, opposite3.12 strand Rgcc regulator of cell cycle 3.13 Dapl1 death associatedprotein-like 1 3.14 Slitrk4 SLIT and NTRK-like family, member 4 3.20Cyp26a1 cytochrome P450, family 26, subfamily a, 3.20 polypeptide 1 Cd96CD96 antigen 3.21 Mrgpra2b MAS-related GPR, member A2B 3.25 Lynx1Ly6/neurotoxin 1 3.25 Pou1f1 POU domain, class 1, transcription factor 13.25 Klrg1 killer cell lectin-like receptor subfamily G, 3.25 member 1S100a9 S100 calcium binding protein A9 3.25 (calgranulin B) Tlr5toll-like receptor 5 3.26 Klra21 killer cell lectin-like receptorsubfamily A, 3.27 member 21 Mrgpra2a MAS-related GPR, member A2A 3.28Ptprcap protein tyrosine phosphatase, receptor 3.29 type, Cpolypeptide-associated protein I830127L07Rik RIKEN cDNA I830127L07 gene3.30 9530053A07Rik RIKEN cDNA 9530053A07 gene 3.31 Olfr288 olfactoryreceptor 288 3.37 Ly6c1 lymphocyte antigen 6 complex, locus C1 3.40 Nkg7natural killer cell group 7 sequence 3.41 Folr4 folate receptor 4(delta) 3.44 Ctsk cathepsin K 3.46 Gpr83 G protein-coupled receptor 833.47 Unc5cl unc-5 homolog C (C. elegans)-like 3.47 Fcer1a Fc receptor,IgE, high affinity I, alpha 3.58 polypeptide Klra7 killer celllectin-like receptor, subfamily A, 3.60 member 7 1810041L15Rik RIKENcDNA 1810041L15 gene 3.62 Nsg2 neuron specific gene family member 2 3.62G0s2 G0/G1 switch gene 2 3.68 Sall3 sal-like 3 (Drosophila) 3.68 S100a8S100 calcium binding protein A8 3.70 (calgranulin A) Klrc1 killer celllectin-like receptor subfamily C, 3.71 member 1 Gm13363 predicted gene13363 3.77 Slc2a13 solute carrier family 2 (facilitated glucose 3.77transporter), member 13 Upp1 uridine phosphorylase 1 3.78 Tnik TRAF2 andNCK interacting kinase 3.82 Klra1 killer cell lectin-like receptor,subfamily A, 3.85 member 1 Tcrd-V1 T cell receptor delta, variable 13.86 Lrrn4 leucine rich repeat neuronal 4 3.89 Mycn v-mycmyelocytomatosis viral related 3.92 oncogene, neuroblastoma derived(avian) Fam155a family with sequence similarity 155, 3.93 member A Myo16myosin XVI 3.96 Xcl1 chemokine (C motif) ligand 1 4.01 Lcn2 lipocalin 24.10 Gzmk granzyme K 4.11 A530013C23Rik RIKEN cDNA A530013C23 gene 4.20Krt75 keratin 75 4.23 1100001G20Rik RIKEN cDNA 1100001G20 gene 4.24Efna2 ephrin A2 4.24 C330013E15Rik RIKEN cDNA C330013E15 gene 4.27 Hecw2HECT, C2 and WW domain containing E3 4.31 ubiquitin protein ligase 2March11 membrane-associated ring finger 4.35 (C3HC4) 11 4930588J15RikRIKEN cDNA 4930588J15 gene 4.37 Sucnr1 succinate receptor 1 4.64 Smok4asperm motility kinase 4A 4.85 I730030J21Rik RIKEN cDNA I730030J21 gene5.14 Slc15a2 solute carrier family 15 (H+/peptide 5.21 transporter),member 2 Penk preproenkephalin 5.25 1700034I23Rik FUN14 domaincontaining 2 pseudogene 5.30 Tnfsf18 tumor necrosis factor (ligand) 5.64superfamily, member 18 Cox6a2 cytochrome c oxidase subunit VIa 5.68polypeptide 2 Csf2 colony stimulating factor 2 (granulocyte- 5.81macrophage) Actg1 actin, gamma, cytoplasmic 1 5.85 Apold1 apolipoproteinL domain containing 1 6.06 Ip6k2 inositol hexaphosphate kinase 2 7.27Baiap3 BAI1-associated protein 3 7.29 Hsd3b1 hydroxy-delta-5-steroiddehydrogenase, 3 9.34 beta- and steroid delta-isomerase 1 Sec14l4SEC14-like 4 (S. cerevisiae) 10.28 Itgad integrin, alpha D 10.29

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

-   Acosta-Alvear, D., Zhou, Y., Blais, A., Tsikitis, M., Lents, N. H.,    Arias, C., Lennon, C. J., Kluger, Y., and Dynlacht, B. D. (2007).    XBP1 controls diverse cell type- and condition-specific    transcriptional regulatory networks. Mol Cell 27, 53-66.-   Barnett, B., Kryczek, I., Cheng, P., Zou, W., and Curiel, T. J.    (2005). Regulatory T cells in ovarian cancer: biology and    therapeutic potential. Am J Reprod Immunol 54, 369-377.-   Bhowmick, N. A., Neilson, E. G., and Moses, H. L. (2004). Stromal    fibroblasts in cancer initiation and progression. Nature 432,    332-337.-   Bollard, C. M., Gottschalk, S., Leen, A. M., Weiss, H.,    Straathof, K. C., Carrum, G., Khalil, M., Wu, M. F., Huls, M. H.,    Chang, C. C., et al. (2007). Complete responses of relapsed lymphoma    following genetic modification of tumor-antigen presenting cells and    T-lymphocyte transfer. Blood 110, 2838-2845.-   Callahan, M. J., Nagymanyoki, Z., Bonome, T., Johnson, M. E.,    Litkouhi, B., Sullivan, E. H., Hirsch, M. S., Matulonis, U. A., Liu,    J., Birrer, M. J., et al. (2008). Increased HLA-DMB expression in    the tumor epithelium is associated with increased CTL infiltration    and improved prognosis in advanced-stage serous ovarian cancer. Clin    Cancer Res 14, 7667-7673.-   Carrasco, D. R., Sukhdeo, K., Protopopova, M., Sinha, R., Enos, M.,    Carrasco, D. E., Zheng, M., Mani, M., Henderson, J., Pinkus, G. S.,    et al. (2007). The differentiation and stress response factor XBP-1    drives multiple myeloma pathogenesis. Cancer Cell 11, 349-360.-   Caton, M. L., Smith-Raska, M. R., and Reizis, B. (2007). Notch-RBP-J    signaling controls the homeostasis of CD8− dendritic cells in the    spleen. J Exp Med 204, 1653-1664.-   Chen, X., Iliopoulos, D., Zhang, Q., Tang, Q., Greenblatt, M. B.,    Hatziapostolou, M., Lim, E., Tam, W. L., Ni, M., Chen, Y., et al.    (2014). XBP1 promotes triple-negative breast cancer by controlling    the HIF1alpha pathway. Nature 508, 103-107.-   Conejo-Garcia, J. R., Benencia, F., Courreges, M. C., Kang, E.,    Mohamed-Hadley, A., Buckanovich, R. J., Holtz, D. O., Jenkins, A.,    Na, H., Zhang, L., et al. (2004).-   Tumor-infiltrating dendritic cell precursors recruited by a    beta-defensin contribute to vasculogenesis under the influence of    Vegf-A. Nat Med 10, 950-958.-   Conejo-Garcia, J. R., Buckanovich, R. J., Benencia, F.,    Courreges, M. C., Rubin, S. C., Carroll, R. G., and Coukos, G.    (2005). Vascular leukocytes contribute to tumor vascularization.    Blood 105, 679-681.-   Coussens, L. M., Tinkle, C. L., Hanahan, D., and Werb, Z. (2000).    MMP-9 supplied by bone marrow-derived cells contributes to skin    carcinogenesis. Cell 103, 481-490.-   Coussens, L. M., and Werb, Z. (2002). Inflammation and cancer.    Nature 420, 860-867.-   Cubillos-Ruiz, J. R., Baird, J. R., Tesone, A. J., Rutkowski, M. R.,    Scarlett, U. K., Camposeco-Jacobs, A. L., Anadon-Arnillas, J.,    Harwood, N. M., Korc, M., Fiering, S. N., et al. (2012).    Reprogramming tumor-associated dendritic cells in vivo using    microRNA mimetics triggers protective immunity against ovarian    cancer. Cancer Res (Published OnlineFirst February 3).-   Cubillos-Ruiz, J. R., Engle, X., Scarlett, U. K., Martinez, D.,    Barber, A., Elgueta, R., Wang, L., Nesbeth, Y., Durant, Y.,    Gewirtz, A. T., et al. (2009). Polyethylenimine-based siRNA    nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to    elicit therapeutic antitumor immunity. J Clin Invest 119, 2231-2244.-   Cubillos-Ruiz, J. R., Rutkowski, M., and Conejo-Garcia, J. R.    (2010). Blocking ovarian cancer progression by targeting tumor    microenvironmental leukocytes. Cell Cycle 9, 260-268.-   Curiel, T. J., Wei, S., Dong, H., Alvarez, X., Cheng, P., Mottram,    P., Krzysiek, R., Knutson, K. L., Daniel, B., Zimmermann, M. C., et    al. (2003). Blockade of B7-H1 improves myeloid dendritic    cell-mediated antitumor immunity. Nat Med 9, 562-567.-   De Palma, M., Murdoch, C., Venneri, M. A., Naldini, L., and    Lewis, C. E. (2007). Tie2-expressing monocytes: regulation of tumor    angiogenesis and therapeutic implications. Trends Immunol 28,    519-524.-   Dinulescu, D. M., Ince, T. A., Quade, B. J., Shafer, S. A., Crowley,    D., and Jacks, T. (2005). Role of K-ras and Pten in the development    of mouse models of endometriosis and endometrioid ovarian cancer.    Nat Med 11, 63-70.-   Dudley, M. E., Wunderlich, J. R., Robbins, P. F., Yang, J. C., Hwu,    P., Schwartzentruber, D. J., Topalian, S. L., Sherry, R.,    Restifo, N. P., Hubicki, A. M., et al. (2002). Cancer regression and    autoimmunity in patients after clonal repopulation with antitumor    lymphocytes. Science 298, 850-854.-   Flesken-Nikitin, A., Choi, K. C., Eng, J. P., Shmidt, E. N., and    Nikitin, A. Y. (2003). Induction of carcinogenesis by concurrent    inactivation of p53 and Rb1 in the mouse ovarian surface epithelium.    Cancer Res 63, 3459-3463.-   Gomez, B. P., Riggins, R. B., Shajahan, A. N., Klimach, U., Wang,    A., Crawford, A. C., Zhu, Y., Zwart, A., Wang, M., and Clarke, R.    (2007). Human X-box binding protein-1 confers both estrogen    independence and antiestrogen resistance in breast cancer cell    lines. FASEB J 21, 4013-4027.-   Hamanishi, J., Mandai, M., Iwasaki, M., Okazaki, T., Tanaka, Y.,    Yamaguchi, K., Higuchi, T., Yagi, H., Takakura, K., Minato, N., et    al. (2007). Programmed cell death 1 ligand 1 and tumor-infiltrating    CD8+ T lymphocytes are prognostic factors of human ovarian cancer.    Proc Natl Acad Sci USA 104, 3360-3365.-   Han, L. Y., Fletcher, M. S., Urbauer, D. L., Mueller, P., Landen, C.    N., Kamat, A. A., Lin, Y. G., Merritt, W. M., Spannuth, W. A.,    Deavers, M. T., et al. (2008). HLA class I antigen processing    machinery component expression and intratumoral T-Cell infiltrate as    independent prognostic markers in ovarian carcinoma. Clin Cancer Res    14, 3372-3379.-   Herber, D. L., Cao, W., Nefedova, Y., Novitskiy, S. V., Nagaraj, S.,    Tyurin, V. A., Corzo, A., Cho, H. I., Celis, E., Lennox, B., et al.    (2010). Lipid accumulation and dendritic cell dysfunction in cancer.    Nat Med 16, 880-886.-   Hetz, C., Chevet, E., and Harding, H. P. (2013). Targeting the    unfolded protein response in disease. Nature reviews Drug discovery    12, 703-719.-   Hollien, J., Lin, J. H., Li, H., Stevens, N., Walter, P., and    Weissman, J. S. (2009). Regulated Irel-dependent decay of messenger    RNAs in mammalian cells. J Cell Biol 186, 323-331.-   Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009). Systematic    and integrative analysis of large gene lists using DAVID    bioinformatics resources. Nature protocols 4, 44-57.-   Huarte, E., Cubillos-Ruiz, J. R., Nesbeth, Y. C., Scarlett, U. K.,    Martinez, D. G., Buckanovich, R. J., Benencia, F., Stan, R. V.,    Keler, T., Sarobe, P., et al. (2008). Depletion of dendritic cells    delays ovarian cancer progression by boosting antitumor immunity.    Cancer Res 68, 7684-7691.-   Iwakoshi, N. N., Pypaert, M., and Glimcher, L. H. (2007). The    transcription factor XBP-1 is essential for the development and    survival of dendritic cells. J Exp Med 204, 2267-2275.-   Jackson, E. L., Willis, N., Mercer, K., Bronson, R. T., Crowley, D.,    Montoya, R., Jacks, T., and Tuveson, D. A. (2001). Analysis of lung    tumor initiation and progression using conditional expression of    oncogenic K-ras. Genes Dev 15, 3243-3248.-   Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van der    Valk, M., and Berns, A. (2001). Synergistic tumor suppressor    activity of BRCA2 and p53 in a conditional mouse model for breast    cancer. Nat Genet 29, 418-425.-   Langmead, B., Trapnell, C., Pop, M., and Salzberg, S. L. (2009).    Ultrafast and memory-efficient alignment of short DNA sequences to    the human genome. Genome biology 10, R25.-   Lee, A. H., Iwakoshi, N. N., Anderson, K. C., and Glimcher, L. H.    (2003a). Proteasome inhibitors disrupt the unfolded protein response    in myeloma cells. Proc Natl Acad Sci USA 100, 9946-9951.-   Lee, A. H., Iwakoshi, N. N., and Glimcher, L. H. (2003b). XBP-1    regulates a subset of endoplasmic reticulum resident chaperone genes    in the unfolded protein response. Mol Cell Biol 23, 7448-7459.-   Lee, A. H., Scapa, E. F., Cohen, D. E., and Glimcher, L. H. (2008).    Regulation of hepatic lipogenesis by the transcription factor XBP1.    Science 320, 1492-1496.-   Leen, A. M., Myers, G. D., Sili, U., Huls, M. H., Weiss, H.,    Leung, K. S., Carrum, G., Krance, R. A., Chang, C. C., Molldrem, J.    J., et al. (2006). Monoculture-derived T lymphocytes specific for    multiple viruses expand and produce clinically relevant effects in    immunocompromised individuals. Nat Med 12, 1160-1166.-   Li, B., and Dewey, C. N. (2011). RSEM: accurate transcript    quantification from RNA-Seq data with or without a reference genome.    BMC bioinformatics 12, 323.-   Mantovani, A., Allavena, P., Sica, A., and Balkwill, F. (2008).    Cancer-related inflammation. Nature 454, 436-444. Martinon, F.,    Chen, X., Lee, A. H., and Glimcher, L. H. (2010). TLR activation of    the transcription factor XBP1 regulates innate immune responses in    macrophages. Nat Immunol 11, 411-418.-   Morgan, R. A., Dudley, M. E., Wunderlich, J. R., Hughes, M. S.,    Yang, J. C., Sherry, R. M., Royal, R. E., Topalian, S. L.,    Kammula, U. S., Restifo, N. P., et al. (2006). Cancer regression in    patients after transfer of genetically engineered lymphocytes.    Science 314, 126-129.-   Nesbeth, Y., Scarlett, U., Cubillos-Ruiz, J., Martinez, D., Engle,    X., Turk, M. J., and Conejo-Garcia, J. R. (2009). CCL5-mediated    endogenous antitumor immunity elicited by adoptively transferred    lymphocytes and dendritic cell depletion. Cancer Res 69, 6331-6338.-   Nesbeth, Y. C., Martinez, D. G., Toraya, S., Scarlett, U. K.,    Cubillos-Ruiz, J. R., Rutkowski, M. R., and Conejo-Garcia, J. R.    (2010). CD4+ T cells elicit host immune responses to MHC class    II-negative ovarian cancer through CCL5 secretion and CD40-mediated    licensing of dendritic cells. J Immunol 184, 5654-5662.-   Piret, J. P., Mottet, D., Raes, M., and Michiels, C. (2002). CoCl2,    a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce    apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci    973, 443-447.-   Ramakrishnan, R., Tyurin, V. A., Veglia, F., Condamine, T.,    Amoscato, A., Mohammadyani, D., Johnson, J. J., Zhang, L. M.,    Klein-Seetharaman, J., Celis, E., et al. (2014). Oxidized lipids    block antigen cross-presentation by dendritic cells in cancer. J    Immunol 192, 2920-2931.-   Reimold, A. M., Iwakoshi, N. N., Manis, J., Vallabhajosyula, P.,    Szomolanyi-Tsuda, E., Gravallese, E. M., Friend, D., Grusby, M. J.,    Alt, F., and Glimcher, L. H. (2001). Plasma cell differentiation    requires the transcription factor XBP-1. Nature 412, 300-307.-   Robinson, M. D., and Oshlack, A. (2010). A scaling normalization    method for differential expression analysis of RNA-seq data. Genome    biology 11, R25.-   Roby, K. F., Taylor, C. C., Sweetwood, J. P., Cheng, Y., Pace, J.    L., Tawfik, O., Persons, D. L., Smith, P. G., and Terranova, P. F.    (2000). Development of a syngeneic mouse model for events related to    ovarian cancer. Carcinogenesis 21, 585-591.-   Sato, E., Olson, S. H., Ahn, J., Bundy, B., Nishikawa, H., Qian, F.,    Jungbluth, A. A., Frosina, D., Gnjatic, S., Ambrosone, C., et al.    (2005). Intraepithelial CD8+ tumor-infiltrating lymphocytes and a    high CD8+/regulatory T cell ratio are associated with favorable    prognosis in ovarian cancer. Proc Natl Acad Sci USA 102,    18538-18543.-   Scarlett, U. K., Cubillos-Ruiz, J. R., Nesbeth, Y. C., Martinez, D.    G., Engle, X., Gewirtz, A. T., Ahonen, C. L., and    Conejo-Garcia, J. R. (2009). In situ stimulation of CD40 and    Toll-like receptor 3 transforms ovarian cancer-infiltrating    dendritic cells from immunosuppressive to immunostimulatory cells.    Cancer Res 69, 7329-7337.-   Scarlett, U. K., Rutkowski, M. R., Rauwerdink, A. M., Fields, J.,    Escovar-Fadul, X., Baird, J., Cubillos-Ruiz, J. R., Jacobs, A. C.,    Gonzalez, J. L., Weaver, J., et al. (2012). Ovarian cancer    progression is controlled by phenotypic changes in dendritic cells.    J Exp Med.-   Singh, S. P., Niemczyk, M., Saini, D., Awasthi, Y. C., Zimniak, L.,    and Zimniak, P. (2008). Role of the electrophilic lipid peroxidation    product 4-hydroxynonenal in the development and maintenance of    obesity in mice. Biochemistry 47, 3900-3911.-   Singh, S. P., Niemczyk, M., Zimniak, L., and Zimniak, P. (2009). Fat    accumulation in Caenorhabditis elegans triggered by the    electrophilic lipid peroxidation product 4-hydroxynonenal (4-HNE).    Aging 1, 68-80.-   So, J. S., Hur, K. Y., Tarrio, M., Ruda, V., Frank-Kamenetsky, M.,    Fitzgerald, K., Koteliansky, V., Lichtman, A. H., Iwawaki, T.,    Glimcher, L. H., et al. (2012). Silencing of lipid metabolism genes    through IRE1alpha-mediated mRNA decay lowers plasma lipids in mice.    Cell Metab 16, 487-499.-   Sriburi, R., Jackowski, S., Mori, K., and Brewer, J. W. (2004).    XBP1: a link between the unfolded protein response, lipid    biosynthesis, and biogenesis of the endoplasmic reticulum. J Cell    Biol 167, 35-41.-   Vladykovskaya, E., Sithu, S. D., Haberzettl, P., Wickramasinghe, N.    S., Merchant, M. L., Hill, B. G., McCracken, J., Agarwal, A.,    Dougherty, S., Gordon, S. A., et al. (2012). Lipid peroxidation    product 4-hydroxy-trans-2-nonenal causes endothelial activation by    inducing endoplasmic reticulum stress. J Biol Chem 287, 11398-11409.-   Whiteside, T. L. (2008). The tumor microenvironment and its role in    promoting tumor growth. Oncogene 27, 5904-5912.-   Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K.    (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response    to ER stress to produce a highly active transcription factor. Cell    107, 881-891.-   Zhang, L., Conejo-Garcia, J. R., Katsaros, D., Gimotty, P. A.,    Massobrio, M., Regnani, G., Makrigiannakis, A., Gray, H.,    Schlienger, K., Liebman, M. N., et al. (2003). Intratumoral T cells,    recurrence, and survival in epithelial ovarian cancer. N Engl J Med    348, 203-213.-   Zou, W. (2005). Immunosuppressive networks in the tumour environment    and their therapeutic relevance. Nat Rev Cancer 5, 263-274.-   Meredith, M. M., Liu, K., Darrasse-Jeze, G., Kamphorst, A. O.,    Schreiber, H. A., Guermonprez, P., Idoyaga, J., Cheong, C., Yao, K.    H., Niec, R. E., et al. (2012). Expression of the zinc finger    transcription factor zDC (Zbtb46, Btbd4) defines the classical    dendritic cell lineage. J Exp Med 209, 1153-1165.-   Piret, J. P., Mottet, D., Raes, M., and Michiels, C. (2002). CoCl2,    a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce    apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci    973, 443-447.-   Satpathy, A. T., Kc, W., Albring, J. C., Edelson, B. T., Kretzer, N.    M., Bhattacharya, D., Murphy, T. L., and Murphy, K. M. (2012).    Zbtb46 expression distinguishes classical dendritic cells and their    committed progenitors from other immune lineages. J Exp Med 209,    1135-1152.-   Schraml, B. U., van Blijswijk, J., Zelenay, S., Whitney, P. G.,    Filby, A., Acton, S. E., Rogers, N. C., Moncaut, N., Carvajal, J.    J., and Reis e Sousa, C. (2013). Genetic tracing via DNGR-1    expression history defines dendritic cells as a hematopoietic    lineage. Cell 154, 843-858.

1. (canceled)
 2. A method for treatment of cancer in a subject in needthereof, the method comprising: contacting antigen presenting cells(APCs) with an inhibitor of IRE1α, wherein the inhibitor of IRE1α ispresent in an amount sufficient to increase antigen presentation in theAPCs; and administering the APCs contacted with the inhibitor of IRE1αto a subject, wherein the subject has a cancer.
 3. The method of claim2, wherein the cancer is a solid cancer.
 4. The method of claim 3,wherein the solid cancer is an ovarian cancer.
 5. The method of claim 3,wherein the solid cancer is an epithelial cancer, a germ cell cancer, ora stromal tumor.
 6. The method of claim 5, wherein the epithelial canceris a carcinoma or an adenocarcinoma.
 7. The method of claim 2, whereinthe APCs are dendritic cells.
 8. The method of claim 7, wherein thedendritic cells are tumor-associated dendritic cells.
 9. The method ofclaim 2, wherein the APCs provided are obtained from the subject. 10.The method of claim 2, wherein the inhibitor of IRE-1α is anIRE-1α-specific antibody or a small molecule inhibitor of IRE-1α. 11.The method of claim 2, wherein the inhibitor of IRE-1α reduces oreliminates IRE-1α mRNA expression in the APCs.
 12. The method of claim2, wherein the inhibitor of IRE-1α reduces or eliminates IRE-1α proteinexpression in the APCs.
 13. The method of claim 2, wherein the inhibitorof IRE-1α is a nucleic acid molecule that is antisense to anIRE-1α-encoding nucleic acid molecule, an IRE-1α shRNA, an IRE-1α siRNA,a microRNA that targets IRE-1α, or a dominant negative IRE-1α molecule.14. The method of claim 2, further comprising administering anadditional therapeutic agent to the subject.
 15. The method of claim 15,wherein the additional therapeutic agent is a chemotherapeutic agent oran antibody.